<?xml version='1.0' encoding='UTF-8'?><xml><records><record><source-app name="HighWire" version="7.x">Drupal-HighWire</source-app><ref-type name="Journal Article">17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Takasaki, Kevin</style></author><author><style face="normal" font="default" size="100%">Abbasi-Asl, Reza</style></author><author><style face="normal" font="default" size="100%">Waters, Jack</style></author></authors><secondary-authors></secondary-authors></contributors><titles><title><style face="normal" font="default" size="100%">Superficial Bound of the Depth Limit of Two-Photon Imaging in Mouse Brain</style></title><secondary-title><style face="normal" font="default" size="100%">eneuro</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2020</style></year><pub-dates><date><style  face="normal" font="default" size="100%">2020-01-01 00:00:00</style></date></pub-dates></dates><elocation-id><style  face="normal" font="default" size="100%">ENEURO.0255-19.2019</style></elocation-id><doi><style  face="normal" font="default" size="100%">10.1523/ENEURO.0255-19.2019</style></doi><volume><style face="normal" font="default" size="100%">7</style></volume><issue><style face="normal" font="default" size="100%">1</style></issue><abstract><style  face="normal" font="default" size="100%">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.</style></abstract></record></records></xml>