Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein (original) (raw)

Abstract

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett B. D., Jetton T. L., Ying G., Magnuson M. A., Piston D. W. Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets. J Biol Chem. 1996 Feb 16;271(7):3647–3651. doi: 10.1074/jbc.271.7.3647. [DOI] [PubMed] [Google Scholar]
  2. Berger I., Elpeleg O. N., Saada A. Lipoamide dehydrogenase activity in lymphocytes. Clin Chim Acta. 1996 Dec 30;256(2):197–201. doi: 10.1016/s0009-8981(96)06420-0. [DOI] [PubMed] [Google Scholar]
  3. Chance B., Lieberman M. 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. 1978 Jan;26(1):111–117. doi: 10.1016/0014-4835(78)90159-8. [DOI] [PubMed] [Google Scholar]
  4. Chance B. Optical method. Annu Rev Biophys Biophys Chem. 1991;20:1–28. doi: 10.1146/annurev.bb.20.060191.000245. [DOI] [PubMed] [Google Scholar]
  5. Chance B., Schoener B., Oshino R., Itshak F., Nakase Y. Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J Biol Chem. 1979 Jun 10;254(11):4764–4771. [PubMed] [Google Scholar]
  6. Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
  7. Dow J. W., Harding N. G., Powell T. Isolated cardiac myocytes. I. Preparation of adult myocytes and their homology with the intact tissue. Cardiovasc Res. 1981 Sep;15(9):483–514. doi: 10.1093/cvr/15.9.483. [DOI] [PubMed] [Google Scholar]
  8. ESTABROOK R. W. Fluorometric measurement of reduced pyridine nucleotide in cellular and subcellular particles. Anal Biochem. 1962 Sep;4:231–245. doi: 10.1016/0003-2697(62)90006-4. [DOI] [PubMed] [Google Scholar]
  9. Eng J., Lynch R. M., Balaban R. S. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys J. 1989 Apr;55(4):621–630. doi: 10.1016/S0006-3495(89)82859-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green D. R., Reed J. C. Mitochondria and apoptosis. Science. 1998 Aug 28;281(5381):1309–1312. doi: 10.1126/science.281.5381.1309. [DOI] [PubMed] [Google Scholar]
  11. Guezennec C. Y., Lienhard F., Louisy F., Renault G., Tusseau M. H., Portero P. In situ NADH laser fluorimetry during muscle contraction in humans. Eur J Appl Physiol Occup Physiol. 1991;63(1):36–42. doi: 10.1007/BF00760798. [DOI] [PubMed] [Google Scholar]
  12. Hall C. L., Kamin H. The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria. J Biol Chem. 1975 May 10;250(9):3476–3486. [PubMed] [Google Scholar]
  13. Hassinen I., Chance B. Oxidation-reduction properties of the mitochondrial flavoprotein chain. Biochem Biophys Res Commun. 1968 Jun 28;31(6):895–900. doi: 10.1016/0006-291x(68)90536-6. [DOI] [PubMed] [Google Scholar]
  14. Kierdaszuk B., Malak H., Gryczynski I., Callis P., Lakowicz J. R. Fluorescence of reduced nicotinamides using one- and two-photon excitation. Biophys Chem. 1996 Nov 29;62(1-3):1–13. doi: 10.1016/s0301-4622(96)02182-5. [DOI] [PubMed] [Google Scholar]
  15. Koke J. R., Wylie W., Wills M. Sensitivity of flavoprotein fluorescence to oxidative state in single isolated heart cells. Cytobios. 1981;32(127-128):139–145. [PubMed] [Google Scholar]
  16. Kunz D., Luley C., Winkler K., Lins H., Kunz W. S. Flow cytometric detection of mitochondrial dysfunction in subpopulations of human mononuclear cells. Anal Biochem. 1997 Mar 15;246(2):218–224. doi: 10.1006/abio.1997.2007. [DOI] [PubMed] [Google Scholar]
  17. Kunz W. S. Evaluation of electron-transfer flavoprotein and alpha-lipoamide dehydrogenase redox states by two-channel fluorimetry and its application to the investigation of beta-oxidation. Biochim Biophys Acta. 1988 Jan 20;932(1):8–16. doi: 10.1016/0005-2728(88)90134-x. [DOI] [PubMed] [Google Scholar]
  18. Kunz W. S., Gellerich F. N. Quantification of the content of fluorescent flavoproteins in mitochondria from liver, kidney cortex, skeletal muscle, and brain. Biochem Med Metab Biol. 1993 Aug;50(1):103–110. doi: 10.1006/bmmb.1993.1051. [DOI] [PubMed] [Google Scholar]
  19. Kunz W. S., Kunz W. Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria. Biochim Biophys Acta. 1985 Sep 6;841(3):237–246. doi: 10.1016/0304-4165(85)90064-9. [DOI] [PubMed] [Google Scholar]
  20. Kunz W. S., Kuznetsov A. V., Winkler K., Gellerich F. N., Neuhof S., Neumann H. W. Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers. Anal Biochem. 1994 Feb 1;216(2):322–327. doi: 10.1006/abio.1994.1048. [DOI] [PubMed] [Google Scholar]
  21. Kunz W. S. Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria. FEBS Lett. 1986 Jan 20;195(1-2):92–96. doi: 10.1016/0014-5793(86)80137-5. [DOI] [PubMed] [Google Scholar]
  22. Kuznetsov A. V., Mayboroda O., Kunz D., Winkler K., Schubert W., Kunz W. S. Functional imaging of mitochondria in saponin-permeabilized mice muscle fibers. J Cell Biol. 1998 Mar 9;140(5):1091–1099. doi: 10.1083/jcb.140.5.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. König K., So P. T., Mantulin W. W., Tromberg B. J., Gratton E. Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J Microsc. 1996 Sep;183(Pt 3):197–204. [PubMed] [Google Scholar]
  24. Maeda-Yorita K., Aki K. Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart. J Biochem. 1984 Sep;96(3):683–690. doi: 10.1093/oxfordjournals.jbchem.a134886. [DOI] [PubMed] [Google Scholar]
  25. Masters B. R. Noninvasive corneal redox fluorometry. Curr Top Eye Res. 1984;4:139–200. [PubMed] [Google Scholar]
  26. Masters B. R., So P. T., Gratton E. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophys J. 1997 Jun;72(6):2405–2412. doi: 10.1016/S0006-3495(97)78886-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Masters B. R., So P. T., Gratton E. Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy. Ann N Y Acad Sci. 1998 Feb 9;838:58–67. doi: 10.1111/j.1749-6632.1998.tb08187.x. [DOI] [PubMed] [Google Scholar]
  28. Nuutinen E. M., Hiltunen J. K., Hassinen I. E. The glutamate dehydrogenase system and the redox state of mitochondrial free nicotinamide adenine dinucleotide in myocardium. FEBS Lett. 1981 Jun 15;128(2):356–360. doi: 10.1016/0014-5793(81)80116-0. [DOI] [PubMed] [Google Scholar]
  29. Pacioretty L. M., Gilmour R. F., Jr Restoration of transient outward current by norepinephrine in cultured canine cardiac myocytes. Am J Physiol. 1998 Nov;275(5 Pt 2):H1599–H1605. doi: 10.1152/ajpheart.1998.275.5.H1599. [DOI] [PubMed] [Google Scholar]
  30. Patel M. S., Vettakkorumakankav N. N., Liu T. C. Dihydrolipoamide dehydrogenase: activity assays. Methods Enzymol. 1995;252:186–195. doi: 10.1016/0076-6879(95)52022-8. [DOI] [PubMed] [Google Scholar]
  31. Patterson G. H., Knobel S. M., Arkhammar P., Thastrup O., Piston D. W. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5203–5207. doi: 10.1073/pnas.090098797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perham R. N. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry. 1991 Sep 3;30(35):8501–8512. doi: 10.1021/bi00099a001. [DOI] [PubMed] [Google Scholar]
  33. Piston D. W., Knobel S. M., Postic C., Shelton K. D., Magnuson M. A. Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion. J Biol Chem. 1999 Jan 8;274(2):1000–1004. doi: 10.1074/jbc.274.2.1000. [DOI] [PubMed] [Google Scholar]
  34. Piston D. W., Masters B. R., Webb W. W. Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy. J Microsc. 1995 Apr;178(Pt 1):20–27. doi: 10.1111/j.1365-2818.1995.tb03576.x. [DOI] [PubMed] [Google Scholar]
  35. Quistorff B., Haselgrove J. C., Chance B. High spatial resolution readout of 3-D metabolic organ structure: an automated, low-temperature redox ratio-scanning instrument. Anal Biochem. 1985 Aug 1;148(2):389–400. doi: 10.1016/0003-2697(85)90244-1. [DOI] [PubMed] [Google Scholar]
  36. Romashko D. N., Marban E., O'Rourke B. Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1618–1623. doi: 10.1073/pnas.95.4.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saks V. A., Veksler V. I., Kuznetsov A. V., Kay L., Sikk P., Tiivel T., Tranqui L., Olivares J., Winkler K., Wiedemann F. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998 Jul;184(1-2):81–100. [PubMed] [Google Scholar]
  38. Scholz R., Thurman R. G., Williamson J. R., Chance B., Bücher T. Flavin and pyridine nucleotide oxidation-reduction changes in perfused rat liver. I. Anoxia and subcellular localization of fluorescent flavoproteins. J Biol Chem. 1969 May 10;244(9):2317–2324. [PubMed] [Google Scholar]
  39. Segretain D., Rambourg A., Clermont Y. Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat. Anat Rec. 1981 Jun;200(2):139–151. doi: 10.1002/ar.1092000204. [DOI] [PubMed] [Google Scholar]
  40. Shiino A., Haida M., Beauvoit B., Chance B. Three-dimensional redox image of the normal gerbil brain. Neuroscience. 1999;91(4):1581–1585. doi: 10.1016/s0306-4522(98)00670-8. [DOI] [PubMed] [Google Scholar]
  41. Shoffner J. M. Oxidative phosphorylation defects and Alzheimer's disease. Neurogenetics. 1997 May;1(1):13–19. doi: 10.1007/s100480050002. [DOI] [PubMed] [Google Scholar]
  42. Voltti H., Hassinen I. E. Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization. J Bioenerg Biomembr. 1978 Apr;10(1-2):45–58. doi: 10.1007/BF00743226. [DOI] [PubMed] [Google Scholar]
  43. Wallace D. C. Mitochondrial diseases in man and mouse. Science. 1999 Mar 5;283(5407):1482–1488. doi: 10.1126/science.283.5407.1482. [DOI] [PubMed] [Google Scholar]
  44. Williams R. M., Webb W. W. Single granule pH cycling in antigen-induced mast cell secretion. J Cell Sci. 2000 Nov;113(Pt 21):3839–3850. doi: 10.1242/jcs.113.21.3839. [DOI] [PubMed] [Google Scholar]
  45. Xu C., Zipfel W., Shear J. B., Williams R. M., Webb W. W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10763–10768. doi: 10.1073/pnas.93.20.10763. [DOI] [PMC free article] [PubMed] [Google Scholar]