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

Ke Wang,Ping Qiu,Shen Tong,Huiping Liang,Yi Pan

doi:10.1364/boe.437482

Abstract

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.

Highlights

Lymphatic vessels are the transporting vessels of the lymphatic system
The indocyanine green (ICG)-contrasted lymphatic vessels can be clearly resolved in 2-photon fluorescence (2PF) images 300 μm below the skin surface
These are the deepest lymphatic vessels, to the best of our knowledge, that can be resolved in the intact mouse skin through Multiphoton microscopy (MPM)

Summary

Introduction

Lymphatic vessels are the transporting vessels of the lymphatic system. They collect extravasated fluid, macromolecules and cells from the interstitium and return them to the venous arm of the circulation [1,2]. Lymphatic vessels are involved in cancer progression, as the cancer cells may enter into them which leads to lymph node metastases. They carry clear lymph and contain few cells, so optical imaging of lymph vessels typically uses exogenous fluorescent dyes to generate image contrast [1]. Among the various optical imaging modalities, single-photon fluorescence imaging has been the most widely used: Sevick and Kwon imaged the deep conducting lymphatic vessels propelling lymph from the inguinal lymph node to the axillary lymph node in mice [3]; Nakajima et al investigated chronic hindlimb lymphedema in mice [4]; Unno et al measured transition times of dye from injection to knees and inguinal region [5]; Sevick and Rasmussen imaged the pumping of deep lymphatic vessels to lymph nodes in humans [6]; John C. For most of the single-photon fluorescence imaging of lymphatic vessels, it only captures the vessels but cannot simultaneously capture other structures next to or even within the vessel

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Conclusion