In vivo three-photon fluorescence imaging of mouse brain vasculature labeled by Evans blue excited at the NIR-III window

Multiphoton fluorescence microscopy (MFM), renowned for its noninvasiveness and high spatiotemporal resolution, is extensively applied in brain structure imaging in vivo. Three-photon fluorescence (3PF) imaging, excited at the NIR-III window, can penetrate the deepest mouse cerebrovascular. Evans blue, a substance known for its low toxicity, high water solubility, and resistance to metabolism, is frequently employed to assess blood-brain barrier (BBB) permeability. However, its suitability for multiphoton fluorescence imaging of mouse cerebrovascular at the NIR-III window in vivo remains unexplored. In this paper, we conduct a comprehensive analysis of the multiphoton excitation and emission characterization of Evans blue when excited at the NIR-III window. Our findings indicate that 1) Evans blue can generate 3PF signals; 2) it exhibits a substantial three-photon action cross-section (<i>ησ₃</i> ) in plasma; 3) its three-photon emission spectrum measured in vivo agrees with that measured in plasma ex vivo. Drawing upon these findings, we successfully demonstrated the application of 3PF imaging of mouse brain vasculature labeled with Evans blue. Notably, the maximum depth of cerebrovascular is 1550 μm beneath the brain surface, spanning the entire gray matter layer and white matter layer and extending into the hippocampus. Evans blue is thus highly ideal for brain cerebrovascular 3PF imaging in vivo.