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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.

Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD