Skip to main content Accessibility help
×
Home

Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

  • Jorge Vergen (a1), Clifford Hecht (a1), Lyandysha V. Zholudeva (a1), Meg M. Marquardt (a1), Richard Hallworth (a2) and Michael G. Nichols (a1)...

Abstract

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Copyright

Corresponding author

Corresponding author. E-mail: mnichols@creighton.edu

References

Hide All
Agronskaia, A.V., Tertoolen, L. & Gerritsen, H.C. (2004). Fast fluorescence lifetime imaging of calcium in living cells. J Biomed Opt 9, 12301237.
An, J., Camara, A.K., Rhodes, S.S., Riess, M.L. & Stowe, D.F. (2005). Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts. Am J Physiol Heart Circ Physiol 288, H2620H2627.
Belke, D.D., Larsen, T.S., Gibbs, E.M. & Severson, D.L. (2000). Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab 279(5), E1104-13.
Bevington, P.R. & Robinson, D.K. (2002). Data Reduction and Error Analysis for the Physical Sciences. New York: McGraw-Hill.
Bird, D.K., Yan, L., Vrotsos, K.M., Eliceiri, K.W., Vaughan, E.M., Keely, P.J., White, J.G. & Ramanujam, N. (2005). Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res 65, 87668773.
Blinova, K., Carroll, S., Bose, S., Smirnov, A.V., Harvey, J.J., Knutson, J.R. & Balaban, R.S. (2005). Distribution of mitochondrial NADH fluorescence lifetimes: Steady-state kinetics of matrix NADH interactions. Biochemistry 44, 25852594.
Blinova, K., Combs, C., Kellman, P. & Balaban, R.S. (2004). Fluctuation analysis of mitochondrial NADH fluorescence signals in confocal and two-photon microscopy images of living cardiac myocytes. J Microsc 213, 7075.
Chance, B. & Baltscheffsky, H. (1958). Respiratory enzymes in oxidative phosphorylation. VII. Binding of intramitochondrial reduced pyridine nucleotide. J Biol Chem 233, 736739.
Chance, B. & Lieberman, M. (1978). Intrinsic fluorescence emission from the cornea at low temperatures: Evidence of mitochondrial signals and their differing redox states in epithelial and endothelial sides. Exp Eye Res 26, 111117.
Chance, B., Oshino, N., Sugano, T. & Mayevsky, A. (1973). Basic principles of tissue oxygen determination from mitochondrial signals. Adv Exp Med Biol 37A, 277292.
Costello, L.C. & Franklin, R.B. (2006). Tumor cell metabolism: The marriage of molecular genetics and proteomics with cellular intermediary metabolism; proceed with caution! Mol Cancer 5, 59.
Evans, N.D., Gnudi, L., Rolinski, O.J., Birch, D.J. & Pickup, J.C. (2005). Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: Potential for non-invasive glucose sensing in diabetes mellitus. J Photochem Photobiol B 80, 122129.
Huang, S., Heikal, A.A. & Webb, W.W. (2002). Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J 82, 28112825.
Indig, G.L., Anderson, G.S., Nichols, M.G., Bartlett, J.A., Mellon, W.S. & Sieber, F. (2000). Effect of molecular structure on the performance of triarylmethane dyes as therapeutic agents for photochemical purging of autologous bone marrow grafts from residual tumor cells. J Pharm Sci 89, 8899.
Kasischke, K.A., Vishwasrao, H.D., Fisher, P.J., Zipfel, W.R. & Webb, W.W. (2004). Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305, 99103.
Lakowicz, J.R., Szmacinski, H., Nowaczyk, K. & Johnson, M.L. (1992). Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci USA 89, 12711275.
Levene, M.J., Dombeck, D.A., Kasischke, K.A., Molloy, R.P. & Webb, W.W. (2004). In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol 91, 19081912.
Lopaschuk, G.D., Folmes, C.D. & Stanley, W.C. (2007). Cardiac energy metabolism in obesity. Circ Res 101(4), 335347.
Mayevsky, A. & Rogatsky, G.G. (2007). Mitochondrial function in vivo evaluated by NADH fluorescence: From animal models to human studies. Am J Physiol Cell Physiol 292, C615C640.
Nichols, M.G., Barth, E.E. & Nichols, J.A. (2005). Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence. Photochem Photobiol 81, 259269.
Nichols, M.G. & Webb, W.W. (1998). Simultaneous imaging of photofrin and NADH autofluorescence in cell monolayers and multicell tumor spheroids. Photochem Photobiol 67S, 95S.
Piston, D.W., Masters, B.R. & Webb, W.W. (1995). Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy. J Microsc 178(Pt 1), 2027.
Rocheleau, J.V., Head, W.S. & Piston, D.W. (2004). Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J Biol Chem 279, 3178031787.
Romashko, D.N., Marban, E. & O'Rourke, B. (1998). Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci USA 95, 16181623.
Scott, T.G., Spencer, R.D., Leonard, N.J. & Weber, G. (1970). Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models. J Am Chem Soc 92, 687695.
Skala, M.C., Riching, K.M., Bird, D.K., Gendron-Fitzpatrick, A., Eickhoff, J., Eliceiri, K.W., Keely, P.J. & Ramanujam, N. (2007a). In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt 12, 024014.
Skala, M.C., Riching, K.M., Gendron-Fitzpatrick, A., Eickhoff, J., Eliceiri, K.W., White, J.G. & Ramanujam, N. (2007b). In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci USA 104, 1949419499.
Tiede, L.M. & Nichols, M.G. (2006). Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy. Photochem Photobiol 82, 656664.
Tiede, L.M., Rocha-Sanchez, S.M., Hallworth, R., Nichols, M.G. & Beisel, K. (2007). Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy. J Biomed Opt 12, 021004.
Vishwasrao, H.D., Heikal, A.A., Kasischke, K.A. & Webb, W.W. (2005). Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J Biol Chem 280, 2511925126.
Wallace, D.C. (2005). A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine. Annu Rev Genet 39, 359407.
Yu, Q. & Heikal, A.A. (2009). Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level. J Photochem Photobiol B 95, 4657.
Zipfel, W.R., Williams, R.M., Christie, R., Nikitin, A.Y., Hyman, B.T. & Webb, W.W. (2003). Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100, 70757080.

Keywords

Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

  • Jorge Vergen (a1), Clifford Hecht (a1), Lyandysha V. Zholudeva (a1), Meg M. Marquardt (a1), Richard Hallworth (a2) and Michael G. Nichols (a1)...

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed