2-photon fluorescence microscopy of brain cells beyond 2 mm.

3-photon microscopy (3 PM) has been the dominant technology for in vivo imaging of deep brain cells down to the hippocampus. However, tissue attenuation and rapid 3 PM signal decay limit the imaging depth to 1.5 mm. Here, we demonstrate non-invasive 2-photon fluorescence (2PF) imaging to a depth of 2 mm in the mouse brain. Excitation in the 1700-nm window minimizes brain attenuation, while 2-photon excitation reduces signal decay compared with other multiphoton modalities. IRDye 800CW 2-DG enables brain-cell labeling and 2PF excitation in this window. Its 2PF emission extends into the NIR-II region, enabling NIR-II 2PF imaging of brain cells. These results establish 2PF microscopy as a promising approach for deeper brain imaging.