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

Abstract

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 ϳ600 m 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 m, 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.

Three-photon imaging of synthetic dyes in deep layers of the neocortex

“…Such studies have proven extremely valuable in unravelling the functional organization of the sensory cortices in a wide variety of mammals such as rodents [4][5] , cats [6][7][8] , ferrets 9-10 and macaques [11][12] . However, due to the limitation of two-photon imaging of neural activity in the brain to within a depth of 450 µm [13][14][15] , these experiments have been largely restricted to neocortical layers 2/3. Therefore, the functional micro-organization of neural networks in cortical layers 4, 5 and 6, or in any area below the neocortex remains elusive.…”

Set lasers to image

“…3P microscopy just ups the ante-3P lasers emit three photons of infrared light with wavelengths between 1300 nm and 1700 nm, which can to penetrate upwards of a millimeter and half. In a mouse, that's the whole depth of its cortex and a little more, and with clearer contrast along the way 1 . "The image quality you get out of three photon microscopes is astounding, even to people who are accustomed to 2 photon images.…”

Improving laser standards for three-photon microscopy

“… 7 Three-photon microscopy builds on the success of two-photon microscopy using longer excitation wavelengths to reduce light scattering and a three-photon absorption process to achieve better nonlinear confinement in biological tissue, resulting in higher signal to background ratio at larger depths. 8 – 12 However, three-photon excitation also poses unique challenges due to its heightened sensitivity to the fidelity of the laser source.…”