Confocal Microscopy of Mitochondrial Function in Living Cells

  • John J. Lemasters
  • Ting Qian
  • Donna R. Trollinger
  • Wayne E. Cascio
  • Hisayuki Ohata
  • Anna-Liisa Nieminen


Confocal microscopy is an essential tool for studying the physiology and pathophysiology of mitochondria within single living cells. As more parameter-indicating fluorophores are discovered, the usefulness of confocal microscopy will only increase. Uniquely, confocal microscopy permits observation of ΔΨ, pH, Ca2+ oxygen free radical generation, and membrane permeability in single mitochondria of living cells. In the future, confocal microscopy should provide new insights concerning the role of mitochondria in health and disease.


Confocal Microscopy Mitochondrial Permeability Transition Axial Resolution Calcein Fluorescence Pinhole Diameter 
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  1. Armstrong, S. C., and Ganote, C. E., 1991, Effects of 2,3-butanedione monoxime (BDM) on contracture and injury of isolated rat myocytes following metabolic inhibition and ischemia, J. Mol. Cell Cardiol. 23:1001–1014.CrossRefPubMedGoogle Scholar
  2. Baker, A. J., Brandes, R., Schreur, J. H., Camacho, S. A., and Weiner, M. W, 1994, Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1, Biophys. J. 67:1646–1654.PubMedGoogle Scholar
  3. Bassani, J.W., Bassani, R.A., and Bers, D.M., 1995, Calibration of indo-1 and resting intracellular [Ca]i in intact rabbit cardiac myocytes, Biophys. J. 68:1453–1460.PubMedGoogle Scholar
  4. Bellomo, G., Vairetti, M., Stivala, L., Mirabelli, P., Richelmi, P., and Orrenius, S., 1992, Demonstration of nuclear compartmentalization of glutathione in hepatocytes, Proc. Nat, Acad. Sci. USA 89:4412–1416.Google Scholar
  5. Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabò, l., and Zoratti, M., 1992, Modulation of the mitochondrial permeability transition pore: Effect of protons and divalent cations, J. Biol. Chem. 267:2934–2939.PubMedGoogle Scholar
  6. Bers, D. M., Bassani, J. W., and Bassani, R. A., 1993, Competition and redistribution among calcium transport systems in rabbit cardiac myocytes, Cardiovasc. Res. 27:1772–1777.PubMedGoogle Scholar
  7. Beyersmann, D., and Hartwig, A., 1992, The genetic toxicology of cobalt, Toxicol. Appl. Pharmacol. 115:137–145.CrossRefPubMedGoogle Scholar
  8. Byrne, A. M., Lemasters, J. J., and Nieminen, A-L., 1999, Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes, Hepatology 29:1523–1531.CrossRefPubMedGoogle Scholar
  9. Cathcart, R., Schwiers, E., and Ames, B. N., 1983, Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay, Anal. Biochem. 134:111–116.CrossRefPubMedGoogle Scholar
  10. Chacon, E., Reece, J. M., Nieminen, A.-L., Zahrebelski, G., Herman, B., and Lemasters, J. J., 1994, Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: A multiparameter digitized confocal microscopic study, Biophys. J. 66:942–952.PubMedGoogle Scholar
  11. Chacon, E., Ohata, H., Harper, I. S., Trollinger, D. R., Herman, B., and Lemasters, J. J., 1996, Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes, FEBS Lett. 382:31–36.CrossRefPubMedGoogle Scholar
  12. Chance, B., Sies, H., and Boveris, A., 1979, Hydroperoxide metabolism in mammalian organs, Physiol. Rev. 59:527–605.PubMedGoogle Scholar
  13. Cheng, H., Lederer, W. J., and Cannell, M. B., 1993, Calcium sparks: Elementary events underlying excitation contraction coupling in heart muscle, Science 262:740–744.PubMedGoogle Scholar
  14. Dawson, T. L., Gores, G. J., Nieminen, A. L., Herman, B., and Lemasters, J. J., 1993, Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am. J. Physiol. 264:C961–C967.PubMedGoogle Scholar
  15. Di Lisa, F., Gambassi, G., Spurgeon, H., and Hansford, R. G., 1993, Intramitochondrial free calcium in cardiac myocytes in relation to dehydrogenase activation, Cardiovasc. Res. 27:1840–1844.PubMedGoogle Scholar
  16. Ehrenberg, B., Montana, V, Wei, M. D., Wuskell, J. P., and Loew, L. M., 1988, Membrane potential can be determined in individual cells from the nemstian distribution of cationic dyes, Biophys. J. 53:785–794.PubMedGoogle Scholar
  17. Emaus, R. K., Grunwald, R., and Lemasters, J. J., 1986, Rhodamine 123 as a probe of transmembrane potential in isolated rat liver mitochondria: Spectral and metabolic properties, Biochem. Biophys. Acta 850:436–448.PubMedGoogle Scholar
  18. Farkas, D. L., Wei, M.-D., Febbroriello, P., Carson, J. H., and Loew, L. M., 1989, Simultaneous imaging of cell and mitochondrial membrane potentials, Biophys. J. 56:1053–1069.PubMedGoogle Scholar
  19. Forman, H. J., and Boveris, A., 1982, Superoxide radical and hydrogen peroxide in mitochondria, Free Radical Biol. 5:65–90.Google Scholar
  20. Gores, G. J., Flarsheim, C. E., Dawson, T. L., Nieminen, A.-L., Herman, B., and Lemasters, J. J., 1989, Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes, Am. J. Physiol. 257:C347–C354.PubMedGoogle Scholar
  21. Griffiths, E. J., and Halestrap, A. P., 1995, Mitochondrial nonspecific pores remain closed during cardiac ischaemia, but open upon reperfusion, Biochem J. 307:93–98.PubMedGoogle Scholar
  22. Griffiths, E. J., Stern, M. D., and Silverman, H. S., 1997, Measurement of mitochondrial calcium in single living cardiomyocytes by selective removal of cytosolic indo 1, Am. J. Physiol. 273:C37–C44.PubMedGoogle Scholar
  23. Grynkiewicz, G., Poenie, M., and Tsien, R. Y., 1985, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260:3440–3450.PubMedGoogle Scholar
  24. Halestrap, A. P., 1991, Calcium-dependent opening of a nonspecific pore in the mitochondrial inner membrane is inhibited at pH values below 7: Implications for the protective effect of low pH against chemical and hypoxic cell damage, Biochem. J. 278:715–719.PubMedGoogle Scholar
  25. Harper, I. S., Bond, J. M., Chacon, E., Reece, J. M., Herman, B., and Lemasters, J. J., 1993, Inhibition of 1 Na+/Ha+ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes, Bas. Res. Cardiol. 88:430–442.Google Scholar
  26. Haugland, R. P., 1996, Handbook of Fluorescent Probes and Research Chemicals, 6th ed., Molecular Probes, Eugene, OR.Google Scholar
  27. Hughes, B. P., and Barritt, G. J., 1989, Inhibition of the liver cell receptor-activated Ca2+ inflow system by metal ion inhibitors of voltage-operated Ca2+ channels but not by other inhibitors of Ca2+ inflow, Biochim. Biophys. Acta 1013:197–205.PubMedGoogle Scholar
  28. Inoué, S., 1995, Foundations of confocal scanned imaging in light microscopy, in Handbook of Biological Confocal Microscopy, 2nd ed. (J. B. Pawley, Ed.), Plenum, New York, pp. 1–17.Google Scholar
  29. Johnson, L. V, Walsh, M. L., Bockus, B. J., and Chen, L. B., 1981, Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy, J. Cell Biol. 88:526–535.CrossRefPubMedGoogle Scholar
  30. Kawanishi, T., Nieminen, A.-L., Herman, B., and Lemasters, J. J., 1991, Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia, J. Biol. Chem. 266:20062–20069.PubMedGoogle Scholar
  31. Keller, H. E., 1995, Objective lenses for confocal microscopy, in Handbook of Biological Confocal Microscopy, 2nd ed. (J. B. Pawley, Ed.), Plenum, New York, pp. 111–126.Google Scholar
  32. Lattanzio, F. A., and Bartschat, D. K., 1991, The effect of pH on rate constants, ion selectivity, and thermodynamic properties of fluorescent calcium and magnesium indicators, Biochem. Biophys. Res. Commun. 177:184–191.CrossRefPubMedGoogle Scholar
  33. Lemasters, J. J., Ji, S., and Thunnan, R. G., 1981, Centrilobular injury following low flow hypoxia in isolated, perfused rat liver, Science 213:661–663.PubMedGoogle Scholar
  34. Lemasters, J. J., Nieminen, A.-L., Qian, T., Trost, L. C., Elmore, S. P., Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A., and Herman, B., 1998, The mitochondrial permeability transition in cell death: A common mechanism in necrosis, apoptosis, and autophagy, Biochim. Biophys. Acta 1366:177–196.PubMedGoogle Scholar
  35. Lemasters, J. J., Trollinger, D. R., Qian, T, Cascio, W. E., and Ohata, H., 1999, Confocal imaging of Ca2+ pH, electrical potential, and membrane permeability in single living cells, in Methods in Enzymology, Green Fluorescent Protein, Vol. 302 (P. M. Conn, Ed.), Academic, New York, pp. 341–358.Google Scholar
  36. Minsky, M., 1961, Microscopy apparatus, U.S. Patent 3,013,467, Dec. 19, 1961 (filed Nov. 7, 1957).Google Scholar
  37. Minta, A., Kao, J. P. Y, and Tsien, R. Y, 1989, Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores, J. Biol. Chem. 264:8171–8178.PubMedGoogle Scholar
  38. Miyata, H., Silverman, H. S., Sollott, S. J., Lakatta, E. G., Stern, M. D., and Hansford, R. G., 1991, Measurement of mitochondrial free Ca2+ contraction in living single rat cardiac myocytes, Am. J. Physiol. 261:H1123–H1134.PubMedGoogle Scholar
  39. Nieminen, A.-L., Dawson, T. L., Gores, G. J., Kawanishi, T., Herman, B., and Lemasters, J. J., 1990a, Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibition, ionophores, and oxidant chemicals, Bio chem. Biophys. Res. Commun. 167:600–606.Google Scholar
  40. Nieminen, A.-L., Gores, G. J., Dawson, T. L., Herman, B., and Lemasters, J. J., 1990b, Mechanisms of toxic injury by HgCl2 in rat hepatocytes studied by multiparameter digitized video microscopy, in Optical Microscopy for Biology (B. Herman and K. Jacobson, Eds.), Alan R. Liss, New York, pp. 323–335.Google Scholar
  41. Nieminen, A.-L., Saylor, A. K., Herman, B., and Lemasters, J. J., 1994, ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition, Am. J. Physiol. 267:C67–C74.PubMedGoogle Scholar
  42. Nieminen, A.-L., Saylor, A. K., Tesfai, S. A., Herman, B., and Lemasters, J. J., 1995, Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide, Biochem. J. 307:99–106.PubMedGoogle Scholar
  43. Nieminen, A.-L., Byrne, A. M., Herman, B., and Lemasters, J. J., 1997, Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species, Am. J. Physiol. 272:C1286–C1294.PubMedGoogle Scholar
  44. Ohata, H., Chacon, E., Tesfai, S. A., Harper, I. S., Herman, B., and Lemasters, J. J., 1998, Mitochondrial transients in cardiac myocytes during the excitation-contraction cycle: Effects of pacing and hormonal stimulation, J. Bioenerg. Biomembr. 30:207–222.CrossRefPubMedGoogle Scholar
  45. Perticarari, S., Presani, G., Mangiarotti, M. A., and Banfi, E., 1991, Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils, Cytometry 12:687–693.CrossRefPubMedGoogle Scholar
  46. Petronilli, V, Miotto, G., Canton, M., Brini, M., Colonna, R., Bernardi, P., and Di Lisa, F., 1999, Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence, Biophys. J. 76:725–734.PubMedGoogle Scholar
  47. Qian, T., Nieminen, A.-L., Herman, B., and Lemasters, J. J., 1997, Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes, Am. J. Physiol. 273:C1783–C1792.PubMedGoogle Scholar
  48. Qian, T., Trost, L. C., and Lemasters, J. J., 1999, Quenching or misalignment? Confocal microscopy of onset of the mitochondrial permeability transition in cultured hepatocytes, Microsc. Microanal. 5 (Suppl. 2): 468–469.Google Scholar
  49. Reers, M., Smiley, S. T., Mottola-Hartshorn, C., Chen, A., Lin, M., and Chen, L. B., 1995, Mitochondrial membrane potential monitored by JC-1 dye, Methods Enzymol. 260:406–417.PubMedGoogle Scholar
  50. Roe, M. W., Lemasters, J. J., and Herman, B., 1990, Assessment of Fura-2 for measurements of cytosolic free calcium, Cell Calcium 11:63–73.CrossRefPubMedGoogle Scholar
  51. Rome, G., Osen, A., and Valet, G., 1988, Dihydrorhodamine 123: A new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes, Naturwissen schaften 75:354.Google Scholar
  52. Rottenberg, H., and Wu, S., 1997, Mitochondrial dysfunction in lymphocytes from old mice: Enhanced activation of the permeability transition, Biochem. Biophys. Res. Commun. 240:68–74.CrossRefPubMedGoogle Scholar
  53. Seghizzi, P., D’Adda, R, Borleri, D., Barbie, R, and Mosconi, G. 1994, Cobalt myocardiopathy: A critical review of literature, Sci. Total Environ. 150:105–109.CrossRefPubMedGoogle Scholar
  54. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G. D., Jr., and Chen, L. B., 1991, Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation, JC-1, Proc. Natl. Acad. Sci. USA. 88:3671–3675.PubMedGoogle Scholar
  55. Trollinger, D. R., Cascio, W. E., and Lemasters, J. J., 1997, Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes, Biochem. Biophys. Res. Commun. 236:738–742.CrossRefPubMedGoogle Scholar
  56. Trollinger, D. R., Cascio, W. E., and Lemasters, J. J., 2000, Mitochondrial calcium transients in adult rabbit cardiac myocytes: Inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca2+-indicating fluorophores, Biophys. J. 79:39–50.PubMedGoogle Scholar
  57. White, J. G., Amos, W. B., and Fordham, M., 1987, An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy, J. Cell Biol. 105:41–48.PubMedGoogle Scholar
  58. Wieder, E. D., Hang, H., and Pox, M. H., 1993, Measurement of intracellular pH using flow cytometry with carboxy-SNARF-1, Cytometry 14:916–921.CrossRefPubMedGoogle Scholar
  59. Zahrebelski, G., Nieminen, A.-L., Al-Ghoul, K., Qian, T, Herman, B., and Lemasters, J. J., 1995, Progression of subcellular changesduring chemical hypoxia to cultured rat hepatocytes: A laserscanning confocal microscopic study, Hepatology 21:1361–1372.CrossRefPubMedGoogle Scholar
  60. Zoratti, M., and Szabó, I., 1995, The mitochondrial permeability transition, Bio chim. Biophys. Acta 1241: 139–176.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • John J. Lemasters
    • 1
  • Ting Qian
    • 1
  • Donna R. Trollinger
    • 2
  • Wayne E. Cascio
    • 3
  • Hisayuki Ohata
    • 4
  • Anna-Liisa Nieminen
    • 5
  1. 1.Department of Cell Biology and AnatomyUniversity of North Carolina at Chapel HillChapel Hill
  2. 2.Department of Molecular and Cell BiologyUniversity of California at DavisDavis
  3. 3.Department of Medicine, School of MedicineUniversity of North Carolina at Chapel HillChapel Hill
  4. 4.Department of Pharmacology, School of Pharmaceutical SciencesShowa UniversityTokyoJapan
  5. 5.Department of Anatomy, School of MedicineCase Western Reserve UniversityCleveland

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