When translating new imaging modalities into the clinic, one can expect new discoveries. Owing to the high photon
count rates, near-infrared fluorescence (NIRF) offers exquisite sensitivity and high temporal resolution that enable new
insights into human disorders. Herein, the physics of NIRF are highlighted and compared to those involved in nuclear
medicine techniques. The initial application of investigational NIRF involving characterization of the lymphatics in
humans and animal models of human disease yields discoveries not otherwise attainable with conventional imaging.