PIs: David Kleinfeld and Zhuhao Wu Project Summary Imaging the molecular constituents of the brain vasculature and lymphatic connectome The intricate connectivity of the brain vasculature that extends into the skull and the meningeal lymphatic vessels are vital in the systematic cerebral blood flow circulation, immunological responses, and waste product removal. However, our understanding of these complex networks remains limited, largely because of the challenges in imaging the complete three-dimensional structure of these structure at high resolution in heterogeneous biological tissues. Recent technical advances on two fronts, one in molecular characterization and labeling, and the other in tissue clearing, led to the discovery of skull-meninge vascular connections. However, wavefront distortion through imperfectly cleared skull and brain tissue severely deteriorate the resolution of imaging at depth. Here we propose to advance sample preparation and imaging technologies that are compatible with labeling and that mitigate the loss in resolution. Our optical methods involve non-linear imaging as well as the use of femtosecond ablation with temporal focussing of bone and soft tissue. This will enable us to obtain diffraction-limited resolution images at the skull/meningeal/brain transition zone. This pilot project will establish a pipeline to image and reconstruct the entire vasculature system and lymphatic system at cellular resolution in the mouse brain. This is a prelude to the long-term goal of imaging and reconstructing similar atlases for all organs in the body.