Metabolic Mapping of MCF10A Human Breast Cells via Multiphoton Fluorescence Lifetime Imaging of the Coenzyme NADH

Bird, Damian; Long, Yan; Vrotsos, Kristin M.; Eliceiri, Kevin W.; Vaughan, Emily M.; Keely, Patricia J.; White, John; Ramanujam, Nirmala

doi:10.1158/0008-5472.can-04-3922

Cited by 353 publications

(430 citation statements)

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“…The lifetimes of free and protein-bound NADH and their relative contributions measured in vivo in normal tissues are consistent with previous work in cell culture (17,18) and in vivo (19). The fluorescence lifetime of protein-bound NADH and its contribution decreased with precancer development in vivo (Fig.…”

Section: Discussionsupporting

confidence: 90%

“…3a), consistent with the results of a previous study (19). Studies have found that the lifetime of free and protein-bound NADH and the relative contribution of protein-bound NADH decrease with hypoxia in cell culture (17)(18)(19) and tissue slices (20). Under hypoxic conditions, cells favor glycolysis over oxidative phosphorylation for ATP production.…”

Section: Discussionsupporting

confidence: 90%

“…The lifetime of Fluoresbrite YG microspheres (Polysciences) were imaged at the beginning of each day of the in vivo experiments to ensure that the lifetime measurements were consistent from day to day. The microsphere lifetime decay curves were fit to a single exponential decay, resulting in a lifetime of 2.19 Ϯ 0.02 ns (n ϭ 12), consistent with previous studies (17,19,39).…”

Section: Dmba-treated Hamster Cheek Pouch Model Of Oral Cancersupporting

confidence: 68%

“…The simulations were carried out on a double-exponential decay curve [ 1 ϭ 300 ps, 2 ϭ 2,000 ps, and ␣ 2 ranges from 27% to 47% in 1% increments, which are reported values for NADH (17,20)]. These curves were fit to a double-exponential model for cases where: (i) 1 was fixed, (ii) 2 was fixed, (iii) 1 and 2 were fixed, and (iv) all were set as free parameters.…”

Section: Quantification Of the Lifetime And Contribution Of Protein-bmentioning

confidence: 99%

“…Previous studies of NADH lifetimes in cell culture (17)(18)(19) and tissue slices (20) have revealed that the fluorescence lifetime of free and protein-bound NADH, and the contribution of protein-bound NADH decrease with hypoxia. The fluorescence lifetime of protein-bound NADH and its relative contribution has also been shown to decrease with lowand high-grade precancers in the hamster cheek pouch model of oral cancer in vivo (19).…”

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In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Skala

Riching

Gendron‐Fitzpatrick

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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.T he electron transport chain is the most efficient means of energy production in cells. The electron transport chain produces energy in the form of ATP by transferring electrons to molecular oxygen. The metabolic coenzymes FAD and NADH are the primary electron acceptor and donor, respectively, in oxidative phosphorylation. Neoplastic cells have an increased metabolic demand relative to normal cells because of rapid cell division (1), and neoplastic metabolism is associated with changes in the relative concentrations of NADH and FAD (2-4). Many enzymes bind to NADH and FAD in the metabolic pathway (5), and, as favored metabolic pathways shift with cancer progression, the distribution of NADH and FAD binding sites also change (6). Thus, metabolic imaging of the relative amount of NADH and FAD, and their microenvironment (such as binding sites and/or the presence of local quenchers) can shed light on metabolic changes associated with carcinogenesis.The metabolic coenzymes NADH and FAD are autofluorescent and can be monitored nondestructively and without exogenous labels, using optical techniques. The most common optical method for metabolic imaging is the ''redox ratio,'' which is the ratio of the fluorescence intensity of FAD and NADH (7). This optical redox ratio provides relative changes in the oxidationreduction state in the cell. The redox ratio is sensitive to changes in the cellular metabolic rate and...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 90%

Section: Dmba-treated Hamster Cheek Pouch Model Of Oral Cancersupporting

confidence: 68%

Section: Quantification Of the Lifetime And Contribution Of Protein-bmentioning

confidence: 99%

mentioning

confidence: 99%

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In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Skala

Riching

Gendron‐Fitzpatrick

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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915

901

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Fluorescence Imaging

2012

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Label‐free Linear and Nonlinear Spectroscopic Imaging in Biology and Medicine

Cicchi

Pavone

2015

The Optics Encyclopedia

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Modern optical and spectroscopic methods offer several label‐free microscopic modalities for improving diagnostic performances on several biological samples, including cells, tissue slices, and fresh biopsies. The relatively recent implementation of multiple linear and nonlinear microspectroscopic techniques in a multimodal approach provides complementary information on the sample under investigation and offers the possibility for integrating morphological with functional‐chemical information in a label‐free manner. In particular, the multimodal approach is crucial for obtaining a morphochemical quantitative analysis of cell cultures and tissue specimens, providing a high‐resolution label‐free support to standard cytological, histological, and immunohistochemical methods. Optical and biophysical researchers successfully have been employing these microspectroscopic techniques for more than a decade, making them increasingly popular among biologists and medical doctors. The label‐free approach also offers the potential for extending the applicability of the methods from ex vivo samples to in vivo examination on both animal models and human subjects, providing new powerful diagnostic tools that could be employed in a clinical setting in the near future.

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Metabolic Mapping of MCF10A Human Breast Cells via Multiphoton Fluorescence Lifetime Imaging of the Coenzyme NADH

Cited by 353 publications

References 28 publications

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Fluorescence Imaging

Label‐free Linear and Nonlinear Spectroscopic Imaging in Biology and Medicine

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