Resolving arteriolar wall structures in mouse brain in vivo with 3-photon microscopy.

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 labelling or imaging technology. Here, we demonstrate that 3-photon microscopy is an ideal solution. In mouse brain in vivo, excited at the 1700-nm window, label-free third-harmonic generation (THG) imaging and 3-photon fluorescence (3PF) imaging with Alexa 633 labelling colocalize and resolve the internal elastic lamina. Furthermore, Alexa Fluor 594-conjugated Wheat Germ Agglutinin (WGA-594) shows time-dependent labelling behavior: As time lapses, WGA- 594 first labels endothelium, and then vascular smooth muscle cells (VSMCs), which are readily captured and resolved with 3PF imaging. Our results show that 3-photon microscopy (3PM), in combination with proper labelling, is a promising technology for investigating the structures of brain arteriolar wall in vivo. This article is protected by copyright. All rights reserved.