In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, three-dimensional functional imaging technique. This study investigates the potential for in vivo pre-cancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic co-factor, NADH. The dimethylbenz[α]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers were imaged using multiphoton FLIM at 780 nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells had short (0.29 ± 0.03 ns) and long lifetime components (2.03 ± 0.06 ns), attributed to free and protein-bound NADH, respectively. Low grade pre-cancers (mild to moderate dysplasia) and high grade pre-cancers (severe dysplasia and carcinoma in situ) were discriminated from normal tissues by their decreased protein-bound NADH lifetime (p