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

J Biomed Opt. Mar-Apr 2007;12(2):024014. doi: 10.1117/1.2717503.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomarkers, Tumor / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Cricetinae
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Mouth Mucosa / metabolism
  • Mouth Mucosa / pathology
  • Mouth Neoplasms / metabolism*
  • Mouth Neoplasms / pathology*
  • NAD / metabolism*
  • Precancerous Conditions / metabolism*
  • Precancerous Conditions / pathology*

Substances

  • Biomarkers, Tumor
  • NAD