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

Two-photon fluorescence microscopy1 enables scientists in various fields including neuroscience2,3, embryology4 and oncology5 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 tissue3 or the insertion of optical probes6,7. 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.