Developing contrast-enhanced optical coherence tomography (OCT) techniques is important for specific imaging of tissue lesions, molecular imaging, cell-tracking, and highly sensitive microangiography and lymphangiography. Multiplexed OCT imaging in the second near-infrared (NIR-II) window is highly desirable since it allows simultaneous imaging and tracking of multiple biological events in high resolution with deeper tissue penetration in vivo. Here we demonstrate that gold nanobipyramids can function as OCT multiplexing contrast agents, allowing high-resolution imaging of two separate lymphatic flows occurring simultaneously from different drainage basins into the same lymph node in a live mouse. Contrast-enhanced multiplexed lymphangiography of a melanoma tumor in vivo shows that the peritumoral lymph flow upstream of the tumor is unidirectional, and tumor is accessible to such flow. Whereas the lymphatic drainage coming out from the tumor is multidirectional. We also demonstrate real-time tracking of the contrast agents draining from a melanoma tumor specifically to the sentinel lymph node of the tumor and the three-dimensional distribution of the contrast agents in the lymph node.

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Optical coherence tomography (OCT) allows label-free, micron-scale 3D imaging of biological tissues’ fine structures with significant depth and large field-of-view. Here we introduce a novel OCT-based neuroimaging setting, accompanied by a feature segmentation algorithm, which enables rapid, accurate, and high-resolution in vivo imaging of 700 μm depth across the mouse cortex. Using a commercial OCT device, we demonstrate 3D reconstruction of microarchitectural elements through a cortical column. Our system is sensitive to structural and cellular changes at micron-scale resolution in vivo, such as those from injury or disease. Therefore, it can serve as a tool to visualize and quantify spatiotemporal brain elasticity patterns. This highly transformative and versatile platform allows accurate investigation of brain cellular architectural changes by quantifying features such as brain cell bodies’ density, volume, and average distance to the nearest cell. Hence, it may assist in longitudinal studies of microstructural tissue alteration in aging, injury, or disease in a living rodent brain.

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