Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo.

Visualization of lymphatic vessels is key to the understanding of their structure, function, and dynamics. Multiphoton microscopy (MPM) is a potential technology for imaging lymphatic vessels, but tissue scattering prevents its deep penetration in skin. Here we demonstrate deep-skin MPM of the lymphatic vessels in mouse hindlimb in vivo, excited at the 1700 nm window. Our results show that with contrast provided by indocyanine green (ICG), 2-photon fluorescence (2PF) imaging enables noninvasive imaging of lymphatic vessels 300 μm below the skin surface, visualizing both its structure and contraction dynamics. Simultaneously acquired second-harmonic generation (SHG) and third-harmonic generation (THG) images visualize the local environment in which the lymphatic vessels reside. After removing the surface skin layer, 2PF and THG imaging visualize finer structures of the lymphatic vessels: most notably, the label-free THG imaging visualizes lymphatic valves and their open-and-close dynamics in real time. MPM excited at the 1700-nm window thus provides a promising technology for the study of lymphatic vessels.