Reproducible Setup for <em>In vivo</em> Two-photon Imaging of the Contractility of Cervical Lymphatic Vessels and Lymph Flow in Mice

The contractility of cLVs is the driving force for lymph flow and a key mechanism underlying brain toxin cleansing. Here, we present an innovative method for in vivo two-photon imaging of the contractility of cLVs and the long-term observation of the removal of red blood cells with lymph flow in cLVs in mice. The components of the setup include a mini-heating pad adapted to the mouse's neck that allows for increasing the window for imaging up to 5 h while preserving the cLV contractility and the lymph flow; humidity control; a protective casing; and a positioning system that allows for maintaining stable optical visualization of cLVs. The method ensures reproducibility and compatibility with other types of two-photon microscopes and a wide range of objectives, maintaining high-quality images with depth up to 500 µm. The protocol includes detailed stages of surgical preparation of cLVs for maintaining the physiological environment, providing the normal cLV contractility.