Superficial Bound of the Depth Limit of Two-Photon Imaging in Mouse Brain

Two-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. Two-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 postulated that the two-photon depth limit, beyond which results become inaccurate, is where in-focus and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ∼600 µm in mouse cortex. Neither the two-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth of two-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous two-photon and three-photon excitation. Two-photon and three-photon results from superficial locations were almost identical. two-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for two-photon excitation. However, the two-photon depth limit and balance depth were at 450 µm, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the two-photon depth limit in mouse visual cortex.