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.

光声 (PA) 成像 (PAI) 是一种新兴的强大工具，用于对体内血液、淋巴管和淋巴结进行无创实时映射，以诊断癌症、淋巴水肿和其他疾病。在不同的 PAI 仪器中，市售的光栅扫描光声细观 (RSOM) (iThera Medical GmbH., Germany) 可用于不同组织的高分辨率成像，具有很高的临床转化潜力。然而，皮肤光散射阻止了比 1-2 mm 更深的血管和节点的映射，这限制了包括 RSOM 在内的 PAI 的诊断价值。在这里，我们证明基于甘油的组织光学清除 (TOC) 通过减少光散射来克服这一挑战，从而提高 RSOM 深度穿透。在体内小鼠肢体的临床前模型中，在 70% 甘油和 30% 超声 (US) 凝胶的混合物上替换传统的声耦合剂（如水），导致组织成像深度增加 1.5-2 倍，血管直径低至 20 μm 的 3D 可视化. 为了区分血液和淋巴网络，我们整合了无标记 PA 血管造影（即, 血管成像), 它使用血红蛋白作为内源性造影剂, 使用基于外源性 PA 造影剂标记淋巴管的 PA 淋巴造影。与成熟的临床淋巴造影类似，造影剂被注射到组织中并在几分钟内被淋巴管吸收，从而提供快速的 RSOM 淋巴造影。此外，作为潜在的低毒药物货物的 PA 对比染料和多层纳米复合材料的共同注射显示出纳米复合材料在前哨淋巴结中的选择性长期积累。总体而言，我们的研究结果为深度和高分辨率 3D PA 血管和淋巴造影以及使用新的 RSOM 和 TOC 方法的 PA 引导的淋巴给药开辟了前景。