Imaging the Deep Spinal Cord Microvascular Structure and Function with High-Speed NIR-II Fluorescence Microscopy.

The spinal cord (SC) is crucial for a myriad of somatosensory, autonomic signal processing, and transductions. Understanding the SC vascular structure and function thus plays an integral part in neuroscience and clinical research. However, the dense layers of myelinated ascending axons on the dorsal side inconveniently grant the SC tissue with high optical scattering property, which significantly hinders the imaging depth of the SC vasculature in vivo. Commonly used antiscattering techniques such as multiphoton fluorescence microscopy have low imaging speed and cannot capture the rapid vascular particle flow without significant motion blur. Here, advantage of the high penetration of near-infrared (NIR)-II fluorescence is taken to demonstrate a deep SC vascular structural image stack up to 350 µm, comparable to two-photon microscopy. Furthermore, the red blood cells are labelled with the clinically approved NIR dye indocyanine. The combination of a fast NIR camera and indocyanine green-red blood cells (RBCs) makes it possible to attain high-speed 100 frame-per-second NIR-II imaging to identify the corresponding changes in RBC velocity during the external hind leg stimulus. For the first time, it is established that the NIR-II region would be a promising spectral window for SC imaging. NIR-II fluorescence microscopy has excellent potential for clinical and basic science research on SC.