A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic and Mitochondrial Activities

  • Ahmed A. Heikal
Part of the Reviews in Fluorescence book series (RFLU, volume 2010)


Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) are electron carriers that play important roles in a wide range of metabolic activities and mitochondrial functions in eukaryotic cells. NADH and FAD are naturally fluorescent with distinct illumination/emission wavelengths for selective detection. Their autofluorescence is also sensitive to protein binding and local environment. As a result, these intracellular coenzymes have potential as intrinsic biomarkers for a noninvasive imaging of metabolic activities and oxidation–reduction reactions in living cells either in vitro, ex vivo, or in vivo. This chapter highlights recent findings of these coenzymes as natural biomarkers of metabolic and mitochondrial activities with an emphasis on a multiparametric imaging approach.


Fluorescence Lifetime Average Lifetime Anisotropy Decay Multiparametric Approach Intracellular NADH 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The author thanks his former student, Dr. Qianru Yu, for her help in obtaining some of the data used in this chapter. Figure 3 is a courtesy of Dr. Karl Kasischke (University of Rochester, School of Medicine and Dentistry, Rochester, NY) and published here with permission. This work was partially supported by the National Institute of Health (AG030949) and the National Science Foundation (MCB0718741). The editorial comments by Dr. Shelley Smith (University of Minnesota-Duluth) are deeply appreciated.


  1. 1.
    Heikal AA (2010) Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Biomark Med 4:241–263PubMedCrossRefGoogle Scholar
  2. 2.
    Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell. Garland Science, New YorkGoogle Scholar
  3. 3.
    Christen Y (2000) Oxidative stress and Alzheimer disease. Am J Clin Nutr 71:621S–629SPubMedGoogle Scholar
  4. 4.
    Jacquard C, Trioulier Y, Cosker F, Escartin C, Bizat N, Hantraye P, Cancela JM, Bonvento G, Brouillet E (2006) Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation. FASEB J 20:1021–1023PubMedCrossRefGoogle Scholar
  5. 5.
    Duchen MR (2004) Mitochondria in health and disease: perspectives on a new mitochondrial biology. Mol Aspects Med 25:365–451PubMedCrossRefGoogle Scholar
  6. 6.
    Duchen MR (2004) Roles of mitochondria in health and disease. Diabetes 53(Suppl 1): S96–S102PubMedCrossRefGoogle Scholar
  7. 7.
    Chance B (1976) Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of Mitochondria. Circ Res 38(5 Suppl 1):I31–I38PubMedGoogle Scholar
  8. 8.
    Chance B, Cohen P, Jobsis F, Schoener B (1962) Intracellular oxidation-reduction states in vivo. Science 137:499–508PubMedCrossRefGoogle Scholar
  9. 9.
    Chance B, Jamieson D, Coles H (1965) Energy-linked pyridine nucleotide reduction: inhibitory effects of hyperbaric oxygen in vitro and in vivo. Nature 206:257–263PubMedCrossRefGoogle Scholar
  10. 10.
    Chance B, Legallais V, Schoener B (1962) Metabolically linked changes in fluorescence emission spectra of cortex of rat brain, kidney and adrenal gland. Nature 195:1073–1075PubMedCrossRefGoogle Scholar
  11. 11.
    Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. II. Difference spectra. J Biol Chem 217:395–407PubMedGoogle Scholar
  12. 12.
    Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J Biol Chem 217:383–393PubMedGoogle Scholar
  13. 13.
    Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217:409–427PubMedGoogle Scholar
  14. 14.
    Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. IV. The respiratory chain. J Biol Chem 217:429–438PubMedGoogle Scholar
  15. 15.
    Chance B, Williams GR, Holmes WF, Higgins J (1955) Respiratory enzymes in oxidative phosphorylation. V. A mechanism for oxidative phosphorylation. J Biol Chem 217:439–451PubMedGoogle Scholar
  16. 16.
    Stryer L (1999) Biochemistry. W. H. Freeman and Company, New YorkGoogle Scholar
  17. 17.
    Ying W (2008) NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal 10:179–206PubMedCrossRefGoogle Scholar
  18. 18.
    Klaidman LK, Leung AC, Adams JD Jr (1995) High-performance liquid chromatography analysis of oxidized and reduced pyridine dinucleotides in specific brain regions. Anal Biochem 228:312–317PubMedCrossRefGoogle Scholar
  19. 19.
    Glassman WS, Steinberg M, Alfano RR (1994) Time resolved and steady state fluorescence spectroscopy from normal and malignant cultured human breast cell lines. Lasers Life Sci 6:91–98Google Scholar
  20. 20.
    Palmer GM, Keely PJ, Breslin TM, Ramanujam N (2003) Autofluorescence spectroscopy of normal and malignant human breast cell lines. Photochem Photobiol 78:462–469PubMedCrossRefGoogle Scholar
  21. 21.
    Kunz WS, Kunz W (1985) Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria. Biochim Biophys Acta 841:237–246PubMedCrossRefGoogle Scholar
  22. 22.
    Huang S, Heikal AA, Webb WW (2002) Two-photon fluorescence spectroscopy and micro­scopy of NAD(P)H and flavoprotein. Biophys J 82:2811–2825PubMedCrossRefGoogle Scholar
  23. 23.
    Benson RC, Meyer RA, Zaruba ME, McKhann GM (1979) Cellular autofluorescence – is it due to flavins? J Histochem Cytochem 27:44–48PubMedCrossRefGoogle Scholar
  24. 24.
    Müller F (1991) Chemistry and biochemistry of flavoenzymes. CRC, Boca Raton, FLGoogle Scholar
  25. 25.
    Rocheleau J, Head WS, Piston D (2003) Two-photon NAD(P)H and one-photon flavoprotein autofluorescence imaging to examine the metabolic mechanisms of pancreatic islet beta-cell function. Microsc Microanal 9:218–219Google Scholar
  26. 26.
    Chorvat D Jr, Kirchnerova J, Cagalinec M, Smolka J, Mateasik A, Chorvatova A (2005) Spectral unmixing of flavin autofluorescence components in cardiac myocytes. Biophys J 89: L55–L57PubMedCrossRefGoogle Scholar
  27. 27.
    Romashko DN, Marban E, O’Rourke B (1998) Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci USA 95:1618–1623PubMedCrossRefGoogle Scholar
  28. 28.
    Modica-Napolitano JS, Singh KK (2004) Mitochondrial dysfunction in cancer. Mitochondrion 4:755–762PubMedCrossRefGoogle Scholar
  29. 29.
    Scheffler IE (1999) Mitochondria. Wiley-Liss, New YorkCrossRefGoogle Scholar
  30. 30.
    Gore M, Ibbott F, Mc IH (1950) The cozymase of mammalian brain. Biochem J 47:121–127PubMedGoogle Scholar
  31. 31.
    Sporty JL, Kabir MM, Turteltaub KW, Ognibene T, Lin SJ, Bench G (2008) Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae. J Sep Sci 31:3202–3211PubMedCrossRefGoogle Scholar
  32. 32.
    Britz-McKibbin P, Markuszewski MJ, Iyanagi T, Matsuda K, Nishioka T, Terabe S (2003) Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection. Anal Biochem 313:89–96PubMedCrossRefGoogle Scholar
  33. 33.
    Uppal A, Gupta PK (2003) Measurements of NADH concentration in normal and malignant human tissues from breast and oral cavity. Biotechnol Appl Biochem 37:45–50PubMedCrossRefGoogle Scholar
  34. 34.
    Giblin FJ, Reddy VN (1980) Pyridine nucleotides in ocular tissues as determined by the cycling assay. Exp Eye Res 31:601–609PubMedCrossRefGoogle Scholar
  35. 35.
    Matsumura H, Miyachi S (1980) Cycling assay for nicotinamide adenine dinucleotides. Methods Enzymol 69:465–470CrossRefGoogle Scholar
  36. 36.
    Umemura K, Kimura H (2005) Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay. Anal Biochem 338:131–135PubMedCrossRefGoogle Scholar
  37. 37.
    Xie W, Xu A, Yeung ES (2009) Determination of NAD(+) and NADH in a single cell under hydrogen peroxide stress by capillary electrophoresis. Anal Chem 81:1280–1284PubMedCrossRefGoogle Scholar
  38. 38.
    Stanley PE (1971) Determination of subpicomole levels of NADH and FMN using bacterial luciferase and the liquid scintillation spectrometer. Anal Biochem 39:441–453PubMedCrossRefGoogle Scholar
  39. 39.
    Jones JB, Song JJ, Hempen PM, Parmigiani G, Hruban RH, Kern SE (2001) Detection of mitochondrial DNA mutations in pancreatic cancer offers a “mass”-ive advantage over detection of nuclear DNA mutations. Cancer Res 61:1299–1304PubMedGoogle Scholar
  40. 40.
    Armstrong JS (2006) Mitochondria: a target for cancer therapy. Br J Phamcol 147:239–248CrossRefGoogle Scholar
  41. 41.
    Flescher E (2007) Jasmonates in cancer therapy. Cancer Lett 245:1–10PubMedCrossRefGoogle Scholar
  42. 42.
    Neuzil J, Dong LF, Ramnathapuram L, Hahn T, Chladova M, Wang XF, Zobalova R, Prochazka L, Gold M, Freeman R, Turanek J, Akporiaye ET, Dyason JC, Ralph SJ (2007) Vitamin E analogues as a novel group of mitocans: anti-cancer agents that act by targeting mitochondria. Mol Aspects Med 28:607–645PubMedCrossRefGoogle Scholar
  43. 43.
    Kagan VE, Bayir A, Bayir H, Stoyanovsky D, Borisenko GG, Tyurina YY, Wipf P, Atkinson J, Greenberger JS, Chapkin RS, Belikova NA (2009) Mitochondria-targeted disruptors and inhibitors of cytochrome c/cardiolipin peroxidase complexes: a new strategy in anti-apoptotic drug discovery. Mol Nutr Food Res 53:104–114PubMedCrossRefGoogle Scholar
  44. 44.
    Szeto HH (2006) Mitochondria-targeted peptide antioxidants: novel neuroprotective agents. AAPS J 8:E521–E531PubMedCrossRefGoogle Scholar
  45. 45.
    Harper JA, Dickinson K, Brand MD (2001) Mitochondrial uncoupling as a target for drug development for the treatment of obesity. Obes Rev 2:255–265PubMedCrossRefGoogle Scholar
  46. 46.
    Villette S, Pigaglio-Deshayes S, Vever-Bizet C, Validire P, Bourg-Heckly G (2006) Ultraviolet-induced autofluorescence characterization of normal and tumoral esophageal epithelium cells with quantitation of NAD(P)H. Photochem Photobiol Sci 5:483–492PubMedCrossRefGoogle Scholar
  47. 47.
    Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, White JG, Ramanujam N (2007) In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci USA 104(49):19494–19499PubMedCrossRefGoogle Scholar
  48. 48.
    Skala MC, Riching KM, Bird DK, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, Keely PJ, Ramanujam N (2007) In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt 12:024014PubMedCrossRefGoogle Scholar
  49. 49.
    Tilton RG, Baier LD, Harlow JE, Smith SR, Ostrow E, Williamson JR (1992) Diabetes-induced glomerular dysfunction: Links to a more reduced cytosolic ratio of NADH/NAD+. Kidney Int 41:778–788PubMedCrossRefGoogle Scholar
  50. 50.
    Dukes ID, McIntyre MS, Mertz RJ, Philipson LH, Roe MW, Spencer B, Worley JF 3rd (1994) Dependence on NADH produced during glycolysis for beta-cell glucose signaling. J Biol Chem 269:10979–10982PubMedGoogle Scholar
  51. 51.
    Ido Y, Kilo C, Williamson JR (1997) Cytosolic NADH/NAD+, free radicals, and vascular dysfunction in early diabetes mellitus. Diabetologia 40:S115–S117PubMedCrossRefGoogle Scholar
  52. 52.
    Rocheleau JV, Head WS, Piston DW (2004) Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J Biol Chem 279:31780–31787PubMedCrossRefGoogle Scholar
  53. 53.
    Eto K, Tsubamoto Y, Terauchi Y, Sugiyama T, Kishimoto T, Takahashi N, Yamauchi N, Kubota N, Murayama S, Aizawa T, Akanuma Y, Aizawa S, Kasai H, Yazaki Y, Kadowaki T (1999) Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion. Science 283:981–985PubMedCrossRefGoogle Scholar
  54. 54.
    Winstead JA, Moss SA (1972) Gamma-irradiated flavin adenine dinucleotide: a D-amino acid oxidase inhibitor. Radiat Res 52:520–527PubMedCrossRefGoogle Scholar
  55. 55.
    Chance B (1954) Spectrophotometry of intracellular respiratory pigments. Science 120: 767–775PubMedCrossRefGoogle Scholar
  56. 56.
    Chorvat DC (2006) Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes. Eur Biophys J 36:73–83PubMedCrossRefGoogle Scholar
  57. 57.
    Xu C, Webb WW (1996) Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J Opt So Am B 13:481–491CrossRefGoogle Scholar
  58. 58.
    Kasischke KA, Vishwasrao HD, Fisher PJ, Zipfel WR, Webb WW (2004) Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305:99–103PubMedCrossRefGoogle Scholar
  59. 59.
    Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100:7075–7080PubMedCrossRefGoogle Scholar
  60. 60.
    Bhawalkar JD, Shih A, Pan SJ, Liou WS, Swiatkiewicz J, Reinhardt BA, Prasad PN, Cheng PC (1996) Two-photon laser scanning fluorescence microscopy-from a fluorophore and specimen perspective. Bioimaging 4:168–178CrossRefGoogle Scholar
  61. 61.
    Centonze VE, White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens that confocal imaging. Biophys J 75:2015–2024PubMedCrossRefGoogle Scholar
  62. 62.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 245:73–76CrossRefGoogle Scholar
  63. 63.
    Masters BR, So PT (2004) Antecedents of two-photon excitation laser scanning microscopy. Microsc Res Tech 63:3–11PubMedCrossRefGoogle Scholar
  64. 64.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377PubMedCrossRefGoogle Scholar
  65. 65.
    Duysens LN, Amesz J (1957) Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region. Biochim Biophys Acta 24:19–26PubMedCrossRefGoogle Scholar
  66. 66.
    Uppal A, Ghosh N, Datta A, Gupta PK (2005) Fluorimetric estimation of the concentration of NADH from human blood samples. Biotechnol Appl Biochem 41:43–47PubMedCrossRefGoogle Scholar
  67. 67.
    Papadopoulos AJ, Zhadin NN, Steinberg ML, Alfano RR (1999) Fluorescence spectroscopy of normal, SV40-transformed human keratinocytes, and carcinoma cells. Cancer Biochem Biophys 17:13–23PubMedGoogle Scholar
  68. 68.
    Croce AC, Ferrigno A, Vairetti M, Bertone R, Freitasa I, Bottirolia G (2004) Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions. Photochem Photobiol Sci 3:920–926PubMedCrossRefGoogle Scholar
  69. 69.
    Ranji M, Kanemoto S, Matsubara M, Grosso MA, Gorman JH 3rd, Gorman RC, Jaggard DL, Chance B (2006) Fluorescence spectroscopy and imaging of myocardial apoptosis. J Biomed Opt 11:064036PubMedCrossRefGoogle Scholar
  70. 70.
    Pogue BW, Pitts JD, Mycek MA, Sloboda RD, Wilmot CM, Brandsema JF, O’Hara JA (2001) In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy. Photochem Photobiol 74:817–824PubMedCrossRefGoogle Scholar
  71. 71.
    Ariola FS, Mudaliar DJ, Walvick RP, Heikal AA (2006) Dynamics imaging of lipid phases and lipid-marker interactions in model biomembranes. Phys Chem Chem Phys 8:4517–4529PubMedCrossRefGoogle Scholar
  72. 72.
    Yu Q, Heikal AA (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 95:46–57PubMedCrossRefGoogle Scholar
  73. 73.
    Mayevsky A (2009) Mitochondrial function and energy metabolism in cancer cells: Past overview and future perspectives. Mitochondrion 9:165–179PubMedCrossRefGoogle Scholar
  74. 74.
    Masters BR, So PTC (2001) Confocal microscopy and multi-photon excitation microscopy of human skin in vivo. Opt Express 8:1–10CrossRefGoogle Scholar
  75. 75.
    Brecht M, Fee MS, Garaschuk O, Helmchen F, Margrie TW, Svoboda K, Osten P (2004) Novel approaches to monitor and manipulate single neurons in vivo. J Neurosci 24:9223–9227PubMedCrossRefGoogle Scholar
  76. 76.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940PubMedCrossRefGoogle Scholar
  77. 77.
    Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ (2004) In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J Neurophysiol 92:3121–3133PubMedCrossRefGoogle Scholar
  78. 78.
    Lin SX, Maxfield FR (2004) Fluorescence imaging in living animals. Focus on uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy. Am J Physiol Cell Physiol 287:C257–C259PubMedCrossRefGoogle Scholar
  79. 79.
    Atkinson RJ, Shorthouse AJ, Hurlstone DP (2007) Novel colorectal endoscopic in vivo imaging and resection practice: A short practice guide for interventional endoscopists. Tech Coloproctol 11:7–16PubMedCrossRefGoogle Scholar
  80. 80.
    Hogan MC, Stary CM, Balaban RS, Combs CA (2005) NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability. J Appl Physiol 98:1420–1426PubMedCrossRefGoogle Scholar
  81. 81.
    Shuttleworth CW, Brennan AM, Connor JA (2003) NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices. J Neurosci 23:3196–3208PubMedGoogle Scholar
  82. 82.
    Reinert KC, Dunbar RL, Gao W, Chen G, Ebner TJ (2004) Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo. J Neurophysiol 92:199–211PubMedCrossRefGoogle Scholar
  83. 83.
    Reinert KC, Gao W, Chen G, Ebner TJ (2007) Flavoprotein autofluorescence imaging in the cerebellar cortex in vivo. J Neurosci Res 85:3221–3232PubMedCrossRefGoogle Scholar
  84. 84.
    Williams RM, Piston DW, Webb WW (1994) Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J 8:804–813PubMedGoogle Scholar
  85. 85.
    Gniadecki R, Thorn T, Vicanova J, Petersen A, Wulf HC (2000) Role of mitochondria in ultraviolet-induced oxidative stress. J Cell Biochem 80:216–222PubMedCrossRefGoogle Scholar
  86. 86.
    Lisby S, Gniadecki R, Wulf HC (2005) UV-induced DNA damage in human keratinocytes: quantitation and correlation with long-term survival. Exp Dermatol 14:349–355PubMedCrossRefGoogle Scholar
  87. 87.
    Nichols MG, Barth EE, Nichols JA (2005) Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence. Photochem Photobiol 81:259–269PubMedCrossRefGoogle Scholar
  88. 88.
    Perriott LM, Kono T, Whitshell RR, Knobel SM, Piston DW, Granner DK, Powers AC, May JM (2001) Gluocose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps. Am J Physiol Endocrinol Metab 281:E72–E80PubMedGoogle Scholar
  89. 89.
    Kable EPW, Kiemer AK (2005) Non-invasive live-cell measurement of changes in macrophage NAD(P)H by two-photon microscopy. Immunol Lett 96:33–38PubMedCrossRefGoogle Scholar
  90. 90.
    Bennett BD, Jetton TL, Ying G, Magnuson MA, Piston DW (1996) Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets. J Biol Chem 271:3647–3651PubMedCrossRefGoogle Scholar
  91. 91.
    Vishwasrao HD, Heikal AA, Kasischke KA, Webb WW (2005) Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J Biol Chem 280:25119–25126PubMedCrossRefGoogle Scholar
  92. 92.
    Tiede LM, Rocha-Sanchez SM, Hallworth R, Nichols MG, Beisel K (2007) Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy. J Biomed Opt 12:021004PubMedCrossRefGoogle Scholar
  93. 93.
    Rothstein EC, Carroll S, Combs CA, Jobsis PD, Balaban RS (2005) Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters. Biophys J 88:2165–2176PubMedCrossRefGoogle Scholar
  94. 94.
    Christie RH, Bacskai BJ, Zipfel WR, Williams RM, Kajdasz ST, Webb WW, Hyman BT (2001) Growth arrest of individual senile plaques in a model of Alzheimer’s disease observed by in vivo multiphoton microscopy. J Neurosci 21:858–864PubMedGoogle Scholar
  95. 95.
    Weber G (1950) Fluorescence of riboflavin and flavin-adenine dinucleotide. Biochem J 47: 114–121PubMedGoogle Scholar
  96. 96.
    Visser AJ (1984) Kinetics of stacking interactions in flavin adenine dinucleotide from time-resolved flavin fluorescence. Photochem Photobiol 40:703–706PubMedCrossRefGoogle Scholar
  97. 97.
    de Kok A, Visser AJ (1987) Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time-resolved flavin fluorescence. FEBS Lett 218:135–138PubMedCrossRefGoogle Scholar
  98. 98.
    Digris AV, Shakoun VV, Novikov EG, van Hoek A, Claiborne A, Visser AJWG (1999) Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy. Eur Biophys J 28:526–531PubMedCrossRefGoogle Scholar
  99. 99.
    Brolin SE, Agren A (1977) Assay of flavin nucleotides in pancreatic islets by a differential fluorimetric technique. Biochem J 163:159–162PubMedGoogle Scholar
  100. 100.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New YorkCrossRefGoogle Scholar
  101. 101.
    O’Connor DV, Phillips D (1984) Time-correlated single-photon counting. Academic Press, LondonGoogle Scholar
  102. 102.
    Becker W (2005) Advanced time-correlated single-photon counting techniques. Springer, New YorkCrossRefGoogle Scholar
  103. 103.
    Davey AM, Walvick RP, Liu Y, Heikal AA, Sheets ED (2007) Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells. Biophys J 92:343–355PubMedCrossRefGoogle Scholar
  104. 104.
    Niesner R, Peker B, Schlüsche P, Gericke K-H (2004) Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence. Chemphyschem 5:1141–1149PubMedCrossRefGoogle Scholar
  105. 105.
    Bird DK, Yan L, Vrotsos KM, Eliceiri KW, Vaughan EM, Keely PJ, White JG, Ramanujam N (2005) Metabolic mapping of MCF 10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res 65:8766–8773PubMedCrossRefGoogle Scholar
  106. 106.
    Yu Q, Proia M, Heikal AA (2008) Integrated biophotonics approach for noninvasive and multiscale studies of biomolecular and cellular biophysics. J Biomed Opt 13:041315PubMedCrossRefGoogle Scholar
  107. 107.
    Bailey MF, Thompson EHZ, Millar DP (2001) Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy. Methods 25:62–77PubMedCrossRefGoogle Scholar
  108. 108.
    Mayevsky A, Rogatsky GG (2007) Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am J Physiol Cell Physiol 292:C615–C640PubMedCrossRefGoogle Scholar
  109. 109.
    Mayevsky A, Barbiro-Michaely E (2009) Use of NADH fluorescence to determine mitochondrial function in vivo. Int J Biochem Cell Biol 41:1977–1988PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistrySwenson College of Science and Engineering, The University of Minnesota DuluthDuluthUSA
  2. 2.Department of Pharmacy Practice and Pharmaceutical SciencesCollege of Pharmacy, The University of Minnesota DuluthDuluthUSA

Personalised recommendations