Significance: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examination of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technology may be broad.
Critical issues: As the number of applications for these fluorescence imaging techniques expand, identifying and characterizing additional intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances.
Future directions: Future work is needed to evaluate whether a combination of different biochemical and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways.
Keywords: FAD; FLIM; NADH; fluorescence; microscopy; redox ratio.