In vivo three-photon microscopy of subcortical structures within an intact mouse brain

Two-photon fluorescence microscopy 1 enables scientists in various fields including neuroscience 2 , 3 , embryology 4 and oncology 5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6 , 7 . Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue. Three-photon microscopy performed at the infrared wavelength of 1,700 nm makes it possible to image hard-to-reach vascular structures and labelled neurons in the hippocampus of a mouse brain.