LAUSR

The brain arteriolar wall is a multilayered structure, whose integrity is of key significance to the brain function. However, resolving these different layers in anmial models in vivo is hampered by the lack of either labeling or imaging technology. Here, we demonstrate that three‐photon microscopy (3PM) is an ideal solution. In mouse brain in vivo, excited at the 1700‐nm window, label‐free third‐harmonic generation imaging and three‐photon fluorescence (3PF) imaging with Alexa 633 labeling colocalize and resolve the internal elastic lamina. Furthermore, Alexa Fluor 594‐conjugated Wheat Germ Agglutinin (WGA‐594) shows time‐dependent labeling behavior. As time lapses, WGA‐594 first labels endothelium, and then vascular smooth muscle cells, which are readily captured and resolved with 3PF imaging. Our results show that 3PM, in combination with proper labeling, is a promising technology for investigating the structures of brain arteriolar wall in vivo.