Three-photon fluorescence imaging (3PF) can provide a significant advantage over two-photon fluorescence imaging in strongly scattering samples such as a mouse brain. The fluorescence from three-photon excitation falls off as 1/z4
, where z is the distance away from the focal plane, while the fluorescence from two-photon excitation falls off as 1/z2
. As a result, three-photon excitation reduces the background from regions away from the focal plane and improves the signal-to-background ratio by orders of magnitude. Please see Three-Photon Fluorescence Microscopy
for additional information.