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

ABSTRACT

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 postulated that the 2-photon depth limit, beyond which results become inaccurate, is where in- 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 2-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth 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- and 3-photon results from superficial locations were almost identical. 2-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for 2-photon excitation. However, the 2-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 2-photon depth limit in mouse visual cortex.

SIGNIFICANCE STATEMENT This study measures the maximum depth in the mouse brain to which it is possible to obtain quantitatively accurate results with 2-photon microscopy, a form of non-linear fluorescence microscopy.