Revealing Lymphatic Vessel Dynamics in Skin Using Long-Wavelength Multiphoton Imaging

Wang K, Pan Y, Tong S, Liang H, Qiu P. Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo. Biomed Opt Express 2021;12(10):6474-6484. doi: 10.1364/BOE.437482.

Background: Lymphatic vessels maintain fluid balance and immune function, but imaging them in skin is limited by tissue scattering. Multiphoton microscopy (MPM) provides high resolution and optical sectioning, yet shorter excitation wavelengths restrict depth. Using the 1700-nm excitation window reduces scattering and allows deeper, clearer imaging of biological structures.

Hypothesis: This study hypothesized that excitation of indocyanine green (ICG)–labeled lymphatic vessels at the 1700-nm window using multiphoton microscopy would enable high-resolution, in vivo imaging of both the structural and dynamic properties of deep lymphatic vessels in mouse skin.

Methods: The authors employed a laser scanning microscope equipped with galvo mirrors, photomultiplier tubes and a water-immersion objective lens to perform two-photon fluorescence (2PF), second-harmonic generation (SHG) and third-harmonic generation (THG) imaging in mouse hindlimb skin. ICG was injected to label lymphatic vessels, and quantum dots were used to distinguish blood vessels. Image acquisition and processing were performed using ScanImage. Noninvasive and exposed-skin preparations were imaged in vivo under isoflurane anesthesia.

Results: Noninvasive 2PF imaging resolved lymphatic vessels up to 300 µm below the skin surface, visualizing vessel morphology and contraction dynamics at an average frequency of 0.8 min^-1. In exposed-skin preparations, THG imaging revealed vessel walls and lymphatic valves, whose opening and closing were recorded in real time. Additionally, micrometer-sized particles flowing and transiently trapped near valves were visualized. Signal-to-background ratios reached up to 197:1, confirming image quality and depth penetration.

Conclusions: MPM excited at the 1700-nm window enables noninvasive, deep-tissue visualization of lymphatic vessels and their dynamics in vivo, providing a powerful tool for studying lymphatic physiology and pathology with subcellular resolution.