title: In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia.
abstract: Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenz[alpha]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells has 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 precancers (mild to moderate dysplasia) and high-grade precancers (severe dysplasia and carcinoma in situ) are discriminated from normal tissues by their decreased protein-bound NADH lifetime (p<0.05). Inhibition of cellular glycolysis and oxidative phosphorylation in cell monolayers produces an increase and decrease, respectively, in the protein-bound NADH lifetime (p<0.05). Results indicate that the decrease in protein-bound NADH lifetime with dysplasia is due to a shift from oxidative phosphorylation to glycolysis, consistent with the predictions of neoplastic metabolism. We demonstrate that multiphoton FLIM is a powerful tool for the noninvasive characterization and detection of epithelial precancers in vivo.
authors.affiliation: Duke University, Department of Biomedical Engineering, Durham, North Carolina 27708, USA.
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Skala
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Riching
authors.forename: Damian K
authors.initials: DK
authors.lastname: Bird
authors.forename: Annette
authors.initials: A
authors.lastname: Gendron-Fitzpatrick
authors.forename: Jens
authors.initials: J
authors.lastname: Eickhoff
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Eliceiri
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Keely
authors.forename: Nirmala
authors.initials: N
authors.lastname: Ramanujam
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009061:Mouth Mucosa / Q000378:metabolism / Q000473:pathology; D009062:Mouth Neoplasms / Q000378:metabolism* / Q000473:pathology*; D009243:NAD / Q000378:metabolism*; D011230:Precancerous Conditions / Q000378:metabolism* / Q000473:pathology*
chemical_list: D014408:Biomarkers, Tumor; D009243:NAD