Abstract
Laser scanning confocal microscopy provides the ability to image submicron sections in living cells and tissues. In conjunction with pH-indicating fluorescent probes, confocal microscopy can be used to visualize the distribution of pH inside living cells. Here, we describe a confocal microscopic technique to image intracellular pH in living cells using SNARF-1, a ratiometric pH-indicating fluorescent probe. SNARF-1 is ester-loaded into the cytosol and mitochondria of adult cardiac myocytes. Using 568-nm excitation, emitted fluorescence longer and shorter than 595-nm are imaged and then ratioed after background subtraction. Ratio values for each pixel are converted to values of pH using a standard curve (lookup table). Images of the intracellular distribution of pH show cytosolic and nuclear areas to have a pH of ∼7.1, but in regions corresponding to mitochondria, pH is 8.0, giving a mitochondrial ΔpH of 0.9. During hypoxia, mitochondrial pH decreases to cytosolic values, signifying the collapse of ΔpH. These results illustrate the ability of laser scanning confocal microscopy to image the intracellular distribution of pH in living cells and to determine mitochondrial ΔpH.
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References
Nicholls DG, Ferguson SJ (2002) Bioenergetics 3. Academic Press, London
Lemasters JJ, Ramshesh VK (2007) Imaging of mitochondrial polarization and depolarization with cationic fluorophores. Methods Cell Biol 80:283–295
Chacon E, Reece JM, Nieminen AL, Zahrebelski G, Herman B, Lemasters JJ (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(4):942–952
Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ (1995) Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 307(Pt 1):99–106
Trollinger DR, Cascio WE, Lemasters JJ (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(3):738–742
Kawanishi T, Nieminen AL, Herman B, Lemasters JJ (1991) Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia. J Biol Chem 266(30):20062–20069
Acknowledgment
This work was supported, in part, by grants 2-R01 DK37034, 1 R01 DK073336, and 1 R01 DK070195 from the National Institutes of Health. Imaging facilities were supported, in part, by NIH Center Grant 1P30 CA138313.
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© 2012 Springer Science+Business Media, LLC
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Ramshesh, V.K., Lemasters, J.J. (2012). Imaging of Mitochondrial pH Using SNARF-1. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 810. Humana Press. https://doi.org/10.1007/978-1-61779-382-0_16
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DOI: https://doi.org/10.1007/978-1-61779-382-0_16
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