Publisher Summary This chapter focuses on the nicotinamide adenine dinucleotide (NADH) imaging of tissues and whole organs. It describes the methodology and instrumentation for in vivo semiquantitative NADH-fluorescence imaging and provides examples of measurements on a beating rat heart in a Langendorff perfusion set-up, of measurements on rat transplant kidneys, and of in vivo measurements on rats. In mitochondria, the major net energy conversion is based on electron transport from reduced NADH to molecular O2 via a series of catabolic reactions in the respiratory chain. NADH is a co-enzyme that carries electrons that are released in the tricarboxylic acid cycle to the respiratory chain. Provided that sufficient O2 is present, NADH donates those electrons to the respiratory chain and gets oxidized to NAD+. The respiratory chain converts the potential energy of the electrons into a proton gradient across the mitochondrial membrane, which drives adenosine triphosphate (ATP) production. The chapter also describes the biomedical and clinical applications of NADH fluorescence imaging. It is expected that the development of methodology and instrumentation for in vivo semiquantitative NADH-fluorescence imaging will further the integration of this optical non-invasive technique in clinical practice.