Advertisement

Time-Resolved Spectrometry of Mitochondrial NAD(P)H Fluorescence and Its Applications for Evaluating the Oxidative State in Living Cells

  • Julia Horilova
  • Hauke Studier
  • Zuzana Nadova
  • Pavol Miskovsky
  • Dusan ChorvatJr.
  • Alzbeta Marcek ChorvatovaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1264)

Abstract

Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence (Chorvat and Chorvatova, Laser Phys Lett 6: 175–193, 2009). Here, we describe the detection and measurement of endogenous mitochondrial NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence directly in living cultured cells using fluorescence lifetime spectrometry imaging after excitation with 405 nm picosecond (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.

Key words

Mitochondrial oxidative state Endogenous NAD(P)H fluorescence FLIM Time-resolved spectrometry Energy metabolism 

Notes

Acknowledgments

Authors acknowledge support from Integrated Initiative of European Laser Research Infrastructures LASERLAB-EUROPE III (grant agreement no 284464, EC’s Seventh Framework Programme), EC’s Seventh Framework Programme CELIM 316310 project, the Research Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA No 1/0296/11, and the Slovak Research and Development Agency under the contract APVV-0242-11.

References

  1. 1.
    Chorvat D Jr, Chorvatova A (2009) Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues. Laser Phys Lett 6:175–193CrossRefGoogle Scholar
  2. 2.
    Becker W (2005) Advanced time-correlated single photon counting techniques. Springer, New York, NYCrossRefGoogle Scholar
  3. 3.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy introducing the phase-modulation methods. Springer, New York, NYCrossRefGoogle Scholar
  4. 4.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York, NYCrossRefGoogle Scholar
  5. 5.
    Chance B, Cohen P, Jobsis F, Schoener B (1962) Intracellular oxidation-reduction states in vivo. Science 137:499–508PubMedCrossRefGoogle Scholar
  6. 6.
    Chance B, Ernster L, Garland PB, Lee CP, Light PA, Ohnishi T, Ragan CI, Wong D (1967) Flavoproteins of the mitochondrial respiratory chain. Proc Natl Acad Sci U S A 57:1498–1505PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Chance B, Nioka S, Warren W, Yurtsever G (2005) Mitochondrial NADH as the bellwether of tissue O2 delivery. Adv Exp Med Biol 566:231–242PubMedCrossRefGoogle Scholar
  8. 8.
    Chorvatova A, Chorvat D Jr (2014) Review of tissue fluorophores and their spectroscopic characteristics. In: Marcu L, French P, Elson D (eds) Fluorescence lifetime spectroscopy and imaging for tissue biomedical diagnostics. CRC Press Publ, Boca Raton, FL, pp 47–84CrossRefGoogle Scholar
  9. 9.
    Tadrous PJ, Siegel J, French PM, Shousha S, Lalani e-N, Stamp GW (2003) Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer. J Pathol 199:309–317PubMedCrossRefGoogle Scholar
  10. 10.
    Berezin MY, Achilefu S (2010) Fluorescence lifetime measurements and biological imaging. Chem Rev 110:2641–2684PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Chorvatova A, Mateasik A, Chorvat D Jr (2013) Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells. Laser Phys Lett 10:125703CrossRefGoogle Scholar
  12. 12.
    Blinova K, Carroll S, Bose S, Smirnov AV, Harvey JJ, Knutson JR, Balaban RS (2005) Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions. Biochemistry 44:2585–2594PubMedCrossRefGoogle Scholar
  13. 13.
    Sud D, Zhong W, Beer DG, Mycek MA (2006) Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models. Opt Express 14:4412–4426PubMedCrossRefGoogle Scholar
  14. 14.
    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
  15. 15.
    Konig K, Riemann I (2003) High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8:432–439PubMedCrossRefGoogle Scholar
  16. 16.
    Jamme F, Kascakova S, Villette S, Allouche F, Pallu S, Rouam V, Refregiers M (2013) Deep UV autofluorescence microscopy for cell biology and tissue histology. Biol Cell 105:277–288PubMedCrossRefGoogle Scholar
  17. 17.
    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
  18. 18.
    Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML (1992) Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A 89:1271–1275PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Chorvatova A, Aneba S, Mateasik A, Chorvat D, Comte B (2013) Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes. J Biomed Opt 18:67009CrossRefGoogle Scholar
  20. 20.
    Romashko DN, Marban E, O’Rourke B (1998) Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci U S A 95:1618–1623PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Feenstra KA (2002) Long term dynamics of proteins and peptides. Ponsen & Looijen, Wageningen, pp 119–143Google Scholar
  22. 22.
    Schneckenburger H, Stock K, Lyttek M, Strauss WS, Sailer R (2004) Fluorescence lifetime imaging (FLIM) of rhodamine 123 in living cells. Photochem Photobiol Sci 3:127–131PubMedCrossRefGoogle Scholar
  23. 23.
    Stringari C, Nourse JL, Flanagan LA, Gratton E (2012) Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential. PLoS One 7:e48014PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Chorvatova A, Mateasik A, Chorvat D Jr (2011) Laser-induced photobleaching of NAD(P)H fluorescence components in cardiac cells resolved by linear unmixing of TCSPC signals. Proc of SPIE 7903:790326-1–790326-9Google Scholar
  25. 25.
    Chorvat D Jr, Abdulla S, Elzwiei F, Mateasik A, Chorvatova A (2008) Screening of cardiomyocyte fluorescence during cell contraction by multi-dimensional TCSPC. Proc SPIE 6860:686029-1–686029-12Google Scholar
  26. 26.
    Warburg O (1956) On the origin of cancer cells. Science 123:309–314PubMedCrossRefGoogle Scholar
  27. 27.
    Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899PubMedCrossRefGoogle Scholar
  28. 28.
    Chorvat D Jr, Chorvatova A (2006) Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes. Eur Biophys J Biophys Lett 36:73–83CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Julia Horilova
    • 1
    • 2
  • Hauke Studier
    • 3
  • Zuzana Nadova
    • 2
    • 4
  • Pavol Miskovsky
    • 2
    • 4
  • Dusan ChorvatJr.
    • 1
  • Alzbeta Marcek Chorvatova
    • 5
    • 1
    Email author
  1. 1.Department of BiophotonicsInternational Laser CenterBratislavaSlovakia
  2. 2.Department of Biophysics, Faculty of SciencePavol Jozef Safarik UniversityKosiceSlovakia
  3. 3.Becker & Hickl GmbHBerlinGermany
  4. 4.Centre for Interdisciplinary Biosciences (CIB), Faculty of SciencePavol Jozef Safarik UniversityKosiceSlovakia
  5. 5.Department of Biotechnology, Faculty of Natural SciencesUniversity of Ss. Cyril and MethodiusTrnavaSlovakia

Personalised recommendations