Endogenous two-photon fluorescence imaging elucidates metabolic changes related to enhanced glycolysis and glutamine consumption in precancerous epithelial tissues.

Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.