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Oxidative phosphorylation pathway in the inner membrane of mitochondria provides the majority of energy needs of eukaryotic cells. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are key metabolic cofactors in energy metabolism and redox reactions in all living cells. The enzymatic activities of these biochemical reactions are dependent on the concentration and distribution of these NADH and FAD coenzymes. Mutations of mitochondrial DNA in diseased cells lead to structural mutations of the electron transport chain complexes (I-IV) and, therefore, numerous health problems such as cancer, neurodegenerative diseases, cardiomyopathy, and ageing. Mitochondria also participate in programmed cell death (apoptosis), generation of free radicals and oxidative stress. As a result, intracellular coenzymes have the potential to serve as natural biomarkers for the respiratory state activities and mitochondria-related health problems. Conventional biochemical methods have provided the bulk information on the concentration of these intrinsic cofactors using cell lysates and, therefore, a snapshot of the enzymatic activities in both normal and diseased cells. These biochemical techniques, however, are incapable of providing the morphological context and molecular conformation dynamics inherent in living cells or tissues. Other steady-state fluorescence-based techniques have been developed since the pioneering work of Britton Chance in the 1960’ s. However, these techniques are limited by the ambiguous distinction between intrinsic NADH concentration and alteration of cellular autofluorescence …