OCT-based label-free in vivo lymphangiography within human skin and areola

Lymphatic system drains lymph fluid from the extracellular space into larger ducts through unidirectional, thin-walled capillaries and collecting vessel network. To transport the fluid, lymphangions, the vascular smooth muscle cells, rhythmically constrict and relax, and valve leaflets open and close in an orchestrated manner to mediate unidirectional flow1. This mechanism is critical for tissue fluid homeostasis and immune cell transport and its dysfunction causes symptoms of edema2. Early detection of dysfunctional lymphatic transport in asymptomatic patients before the onset of symptoms could enable earlier diagnoses and more effective treatments. In addition, the identification of factors that affect lymphatic pumping is an active area of research that will contribute to the development of new pharmacologic strategies to correct lymphatic insufficiency. Most of the current and emerging lymphatic imaging approaches require the administration of exogenous contrast agents either directly into lymphatics via the cannulation of a lymphatic vessel or indirectly into lymphatic plexus via intradermal injection. Unfortunately, most of the existing contrast agents can be toxic and induce side effects to patients3. Moreover, locating and cannulating a lymphatic vessel can be significantly invasive for patients and very difficult in preclinical research using transgenic mouse models due to their small size4. X-ray5, magnetic resonance imaging6, near infrared fluorescence imaging7 and lymphoscintigraphy8 techniques provide a body imaging, albeit with poor resolution, so they are used for imaging the larger vessels and the lymph nodes. On the other hand, a microscope based imaging technique, fluorescence microlymphography9, is limited with its penetrating depth (~200 μm) in tissue but can provide relatively higher resolution (~50 μm), hence it is used only in the visualization of initial lymphatics near injection site. Despite its prominent role in healthy and pathological processes, there are only few methods reported in the literature10,11,12 for the purpose of non-invasively imaging the response of lymphatics within in vivo animal models of disease. So far, none of these techniques has shown its potential in clinical investigations. Optical coherence tomography (OCT) is an emerging tool for non-invasive, label-free in vivo imaging of tissue13. It can provide a better penetration depth in tissue than a microscope (~up to 2 mm) with a high resolution (~10 μm). Optical microangiography (OMAG) is a label-free non-invasive imaging and processing method to obtain 3D in vivo blood perfusion map in tissue beds down to capillary level using Fourier domain OCT14,15. OMAG has been applied to visualize microvasculature in various living tissue including healthy16,17 and pathological human skin18, and mouse cerebral cortex19,20. Previously, label-free imaging of lymphatic vessels has been demonstrated on an in vivo mouse ear model using OCT11,12,21. The difference in optical scattering between lymph fluid, which is nearly transparent, and tissue, which is highly scattering is used as a contrast mechanism. Consequently, the lymphatic vessels appear as low-scattering (darker) regions in the OCT images. Three-dimensional connectivity between vessels and characteristic lymphangion morphology are used for the identification of lymphatic network. The result of lymphangiography maps were confirmed by intra-dermal injection of Evan’s blue dye and monitoring the uptake pathway by surrounding lymph vessels into the lymph nodes11,12. The lymphatic vessels have been visualized by applying a threshold to the OCT intensity images21, or using vesselness models based on Hessian multi-scale filters12. These methods work well on in vivo mouse ear models, however, they have not been able to apply to human skin due to obvious challenges facing optical imaging, i.e., the thicker and higher scattering nature of human skin compared to mouse ear skin. The contrast of the dark areas to the surrounding tissue becomes worse and segmentation algorithms fail to provide a clear separation of these areas from the surrounding tissue in OCT images of human skin. We developed a method to contrast lymphatic vessels within highly scattering tissue by reducing light attenuation effect on OCT cross-sectional images and extracting lymphatic vessels through re-arranged enface OCT images (Supplementary Data). With this method, we are able to non-invasively visualize lymphatic networks along with microvasculature within human skin and areola, without using contrast agents. To our knowledge, this is the first demonstration of OCT based label-free optical lymphangiography (OLAG) within human skin and areola in vivo. Moreover, lymphatic system’s response to inflammation is monitored on an acne lesion within human skin for 7 days. Results showed that OLAG is promising to provide a remarkable alternative tool in the investigation and treatment of pathologic mechanisms involving lymphatic system without the use of exogenous contrast agents on patients.