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Results: 11 to 13 of 13

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In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia.
Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, White JG, Ramanujam N
(2007) Proc Natl Acad Sci U S A 104: 19494-9
MeSH Terms: Animals, Carcinoma, Cell Nucleus, Cricetinae, Cytoplasm, Flavin-Adenine Dinucleotide, Fluorescence, Microscopy, Fluorescence, Multiphoton, Mouth Neoplasms, NAD, Oxidation-Reduction, Precancerous Conditions, Tumor Cells, Cultured
Show Abstract · Added March 11, 2014
Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low- and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.
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13 MeSH Terms
In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia.
Skala MC, Riching KM, Bird DK, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, Keely PJ, Ramanujam N
(2007) J Biomed Opt 12: 024014
MeSH Terms: Animals, Biomarkers, Tumor, Cell Line, Cell Line, Tumor, Cricetinae, Microscopy, Fluorescence, Multiphoton, Mouth Mucosa, Mouth Neoplasms, NAD, Precancerous Conditions
Show Abstract · Added March 11, 2014
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.
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10 MeSH Terms
Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues.
Skala MC, Squirrell JM, Vrotsos KM, Eickhoff JC, Gendron-Fitzpatrick A, Eliceiri KW, Ramanujam N
(2005) Cancer Res 65: 1180-6
MeSH Terms: 9,10-Dimethyl-1,2-benzanthracene, Animals, Carcinogens, Carcinoma in Situ, Carcinoma, Squamous Cell, Cheek, Cricetinae, Epithelial Cells, Fluorescence, Keratins, Male, Mesocricetus, Microscopy, Confocal, Microscopy, Fluorescence, Multiphoton, Mouth Neoplasms, Precancerous Conditions
Show Abstract · Added March 11, 2014
This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.
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16 MeSH Terms