Photoacoustic (PA) imaging (PAI) is an emerging powerful tool for noninvasive real-time mapping of blood and lymphatic vessels and lymph nodes in vivo to diagnose cancer, lymphedema and other diseases. Among different PAI instruments, commercially available raster-scanning optoacoustic mesoscopy (RSOM) (iThera Medical GmbH., Germany) is useful for high-resolution imaging of different tissues with high potential of clinical translation. However, skin light scattering prevents mapping vessels and nodes deeper than 1-2 mm, that limits diagnostic values of PAI including RSOM. Here we demonstrate that glycerol-based tissue optical clearing (TOC) overcomes this challenge by reducing light scattering that improves RSOM depth penetration. In preclinical model of mouse limb in vivo, the replacement of conventional acoustic coupling agents such as water on the mixture of 70 % glycerol and 30 % ultrasound (US) gel resulted in the increase of tissue imaging depth in 1.5-2 times with 3D visualization of vessels with diameter down to 20 μm. To distinguish blood and lymphatic networks, we integrated label-free PA angiography (i.e., imaging of blood vessels), which uses hemoglobin as endogenous contrast agent, with PA lymphography based on labeling of lymphatic vessels with exogenous PA contrast agents. Similar to well-established clinical lymphography, contrast agents were injected in tissue and taken up by lymphatic vessels within a few minutes that provided quick RSOM lymphography. Furthermore, co-injection of PA contrast dye and multilayer nanocomposites as potential low-toxic drug-cargo showed selective prolonged accumulation of nanocomposites in sentinel lymph nodes. Overall, our findings open perspectives for deep and high resolution 3D PA angio- and lymphography, and for PA-guided lymphatic drug delivery using new RSOM & TOC approach.

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