Metabolism plays a critical role in the function of cells and tissues. Changes in metabolic function are hallmarks of numerous conditions, including aging, cancer, obesity and neurodegenerative diseases. Such changes are highly dynamic and heterogeneous at the microscopic scale. Label-free, two-photon microscopic imaging offers opportunities to monitor and characterize such aspects of metabolic function non-destructively. Intensity and lifetime fluorescence measurements from endogenous NAD(P)H and FAD can serve as sensitive indicators of metabolic changes associated with redox state and mitochondrial organization. In fact, a combination of optical metabolic readouts including the redox ratio, NADH bound fraction, and mitochondrial clustering can provide important insights on the specific nature of the metabolic pathway perturbation that yielded the optical changes. Assessment of such information has the potential to improve our understanding, detection, and treatment of numerous diseases. For example, our studies highlight that the use of multiple optical metabolic readouts enables sensitive and specific detection of changes that are associated with adipose tissue type (brown vs beige vs white) and responses to stimuli. In addition, we have discovered that our ability to characterize depth-dependent variations within epithelial tissues, such as the skin and cervix, plays a key role in identifying alterations that occur at the onset of cancer and may be used to develop improved, non-invasive methods for cancer diagnosis. Monitoring of immune system cell activation is another important application area. Such findings pave the way for exploiting label-free, metabolic imaging techniques to understand dynamic cell interaction and their role in the development and treatment of a number of human diseases.