Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX

Nature Methods volume 3, pages 939–945 (2006)Cite this article

Abstract

Molecular oxygen is the primary oxidant in biological systems. The ultimate destination of oxygen in vivo is the mitochondria where it is used in oxidative phosphorylation. The ability of this process to produce an amount of high-energy phosphates adequate to sustain life highly depends on the available amount of oxygen. Despite a vast array of techniques to measure oxygen, major (patho)physiological questions remain unanswered because of the unavailability of quantitative techniques to measure mitochondrial oxygen in situ. Here we demonstrate that mitochondrial PO2 can be directly measured in living cells by harnessing the delayed fluorescence of endogenous protoporphyrin IX (PpIX), thereby providing a technique with the potential for a wide variety of applications. We applied this technique to different cell lines (V-79 Chinese hamster lung fibroblasts, HeLa cells and IMR 32-K1 neuroblastoma cells) and present the first direct measurements of the oxygen gradient between the mitochondria and the extracellular volume.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectra of prompt and delayed luminescence of PpIX.
Figure 2: In vitro calibration experiments.
Figure 3: Fluorescence microscopy.
Figure 4: Calibration of the mitochondrial signal.
Figure 5: PO2 difference between the mitochondria and the extracellular medium.

Similar content being viewed by others

References

  1. Mitchell, P. & Moyle, J. Chemiosmotic hypothesis of oxidative phosphorylation. Nature 213, 137–139 (1967).

    Article  CAS  Google Scholar 

  2. Droge, W. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95 (2002).

    Article  CAS  Google Scholar 

  3. Xi, Q., Cheranov, S.Y. & Jaggar, J.H. Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks. Circ. Res. 97, 354–362 (2005).

    Article  CAS  Google Scholar 

  4. Swartz, H.M. Measuring real levels of oxygen in vivo: opportunities and challenges. Biochem. Soc. Trans. 30, 248–252 (2002).

    Article  CAS  Google Scholar 

  5. Vanderkooi, J.M., Erecinska, M. & Silver, I.A. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am. J. Physiol. 260, C1131–C1150 (1991).

    Article  CAS  Google Scholar 

  6. Hogan, M.C. Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers. J. Appl. Physiol. 86, 720–724 (1999).

    Article  CAS  Google Scholar 

  7. Takahashi, E., Sato, K., Endoh, H., Xu, Z.L. & Doi, K. Direct observation of radial intracellular PO2 gradients in a single cardiomyocyte of the rat. Am. J. Physiol. 275, H225–H233 (1998).

    CAS  PubMed  Google Scholar 

  8. Vanderkooi, J.M., Maniara, G., Green, T.J. & Wilson, D.F. An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem. 262, 5476–5482 (1987).

    CAS  PubMed  Google Scholar 

  9. Koo, Y.E. et al. Real-time measurements of dissolved oxygen inside live cells by organically modified silicate fluorescent nanosensors. Anal. Chem. 76, 2498–2505 (2004).

    Article  CAS  Google Scholar 

  10. Poulson, R. The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria. J. Biol. Chem. 251, 3730–3733 (1976).

    CAS  PubMed  Google Scholar 

  11. Fukuda, H., Casas, A. & Batlle, A. Aminolevulinic acid: from its unique biological function to its star role in photodynamic therapy. Int. J. Biochem. Cell Biol. 37, 272–276 (2005).

    Article  CAS  Google Scholar 

  12. Dalton, J., McAuliffe, C.A. & Slater, D.H. Reaction between molecular oxygen and photo-excited protoporphyrin IX. Nature 235, 388 (1972).

    Article  CAS  Google Scholar 

  13. Chantrell, S.J., McAuliffe, C.A., Munn, R.W. & Pratt, A.C. Excited states of protoporphyrin IX dimethyl ester: reaction of the triplet with carotenoids. J. C. S. Faraday I 60, 858–865 (1977).

    Article  Google Scholar 

  14. Calvo, M.R. et al. Photophysics of protoporphyrin ions in vacuo: triplet-state lifetimes and quantum yields. J. Chem. Phys. 120, 5067–5072 (2004).

    Article  CAS  Google Scholar 

  15. Lo, L.W., Koch, C.J. & Wilson, D.F. Calibration of oxygen-dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. Anal. Biochem. 236, 153–160 (1996).

    Article  CAS  Google Scholar 

  16. Mik, E.G., Donkersloot, C., Raat, N.J. & Ince, C. Excitation pulse deconvolution in luminescence lifetime analysis for oxygen measurements in vivo. Photochem. Photobiol. 76, 12–21 (2002).

    Article  CAS  Google Scholar 

  17. Sudha, B.P., Dixit, N., Moy, V.T. & Vanderkooi, J.M. Reactions of excited-state cytochrome c derivatives. Delayed fluorescence and phosphorescence of zinc, tin, and metal-free cytochrome c at room temperature. Biochemistry 23, 2103–2107 (1984).

    Article  Google Scholar 

  18. Parker, C.A. & Joyce, T.A. Delayed fluorescence and some properties of the chlorophyll triplets. Photochem. Photobiol. 6, 395–406 (1967).

    Article  CAS  Google Scholar 

  19. Dunphy, I., Vinogradov, S.A. & Wilson, D.F. Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence. Anal. Biochem. 310, 191–198 (2002).

    Article  CAS  Google Scholar 

  20. Gardner, L.C., Smith, S.J. & Cox, T.M. Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man. J. Biol. Chem. 266, 22010–22018 (1991).

    CAS  PubMed  Google Scholar 

  21. Kelty, C.J., Brown, N.J., Reed, M.W. & Ackroyd, R. The use of 5-aminolaevulinic acid as a photosensitiser in photodynamic therapy and photodiagnosis. Photochem. Photobiol. Sci. 1, 158–168 (2002).

    Article  CAS  Google Scholar 

  22. Peng, Q. et al. 5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges. Cancer 79, 2282–2308 (1997).

    Article  CAS  Google Scholar 

  23. Morgan, J. & Oseroff, A.R. Mitochondria-based photodynamic anti-cancer therapy. Adv. Drug Deliv. Rev. 49, 71–86 (2001).

    Article  CAS  Google Scholar 

  24. Wilson, B.C., Olivo, M. & Singh, G. Subcellular localization of Photofrin and aminolevulinic acid and photodynamic cross-resistance in vitro in radiation-induced fibrosarcoma cells sensitive or resistant to photofrin-mediated photodynamic therapy. Photochem. Photobiol. 65, 166–176 (1997).

    Article  CAS  Google Scholar 

  25. Wu, S.M., Ren, Q.G., Zhou, M.O., Peng, Q. & Chen, J.Y. Protoporphyrin IX production and its photodynamic effects on glioma cells, neuroblastoma cells and normal cerebellar granule cells in vitro with 5-aminolevulinic acid and its hexylester. Cancer Lett. 200, 123–131 (2003).

    Article  CAS  Google Scholar 

  26. Gaullier, J.M. et al. Subcellular localization of and photosensitization by protoporphyrin IXhuman keratinocytes and fibroblasts cultivated with 5-aminolevulinic acid. Photochem. Photobiol. 62, 114–122 (1995).

    Article  CAS  Google Scholar 

  27. Clark, A., Jr., Clark, P.A., Connett, R.J., Gayeski, T.E. & Honig, C.R. How large is the drop in PO2 between cytosol and mitochondrion? Am. J. Physiol. 252, C583–C587 (1987).

    Article  Google Scholar 

  28. Robiolio, M., Rumsey, W.L. & Wilson, D.F. Oxygen diffusion and mitochondrial respiration in neuroblastoma cells. Am. J. Physiol. 256, C1207–C1213 (1989).

    Article  CAS  Google Scholar 

  29. Rick, K. et al. Pharmacokinetics of 5-aminolevulinic acid-induced protoporphyrin IX in skin and blood. J. Photochem. Photobiol. B 40, 313–319 (1997).

    Article  CAS  Google Scholar 

  30. Van den Boogert, J. et al. 5-Aminolaevulinic acid-induced protoporphyrin IX accumulation in tissues: pharmacokinetics after oral or intravenous administration. J. Photochem. Photobiol. B 44, 29–38 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was in part supported by the Technological Collaboration Grant (TSGE 1048) of the Dutch Ministry of Economic Affairs. As part of this collaboration, K. Boller (Department of Laser Physics, University of Twente, Enschede, The Netherlands) kindly provided the pulsed laser system and tunable optical parametric oscillator. A. van Kuilenburg (Laboratory for Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands) kindly provided IMR 32-K1 Neuroblastoma cells. J.S. and J.A.A. were funded in part by a grant from the Dutch Cancer Society (KWF). The authors thank C. van Oven, K. Pos and P. Goedhart for technical assistance.

Author information

Authors and Affiliations

  1. Department of Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, 1105 AZ, The Netherlands

    Egbert G Mik & Can Ince

  2. Department of Anesthesiology, Erasmus Medical Center, University of Rotterdam, Dr. Molewaterplein 40, Rotterdam, 3015 GD, The Netherlands

    Egbert G Mik

  3. Department of Cell Biology and Histology, Center for Microscopical Research, Academic Medical Center, University of Amsterdam, Amsterdam, 1105 AZ, The Netherlands

    Jan Stap & Jacob A Aten

  4. Laser Center, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, 1105 AZ, The Netherlands

    Michiel Sinaasappel, Johan F Beek & Ton G van Leeuwen

  5. Biophysical Engineering, Biomedical Technology Institute, University of Twente, PO Box 217, Enschede, 7500 AE, The Netherlands

    Ton G van Leeuwen

Authors
  1. Egbert G Mik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan Stap
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michiel Sinaasappel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johan F Beek
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jacob A Aten
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ton G van Leeuwen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Can Ince
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.G.M. conceived and designed the study, performed experiments and wrote the manuscript; J.S. cultured the cells and performed fluorescence microscopy; M.S. contributed to the biological idea; J.F.B. and T.G.L. were involved in the construction of the delayed fluorescence setup; J.A.A. advised on biological experiments; C.I. facilitated the study and supervised the work.

Corresponding author

Correspondence to Egbert G Mik.

Ethics declarations

Competing interests

The authors' institution (The Academic Medical Center in Amsterdam, the Netherlands) has filed a European patent application (number 05076565.0), which covers some of the work described in this article.

Supplementary information

Supplementary Fig. 1

Extended calibration curve in HeLa cells. (PDF 26 kb)

Supplementary Fig. 2

Simultaneous delayed fluorescence and phosphorescence lifetime measurements in a homogenous solution. (PDF 84 kb)

Supplementary Methods (PDF 37 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mik, E., Stap, J., Sinaasappel, M. et al. Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX. Nat Methods 3, 939–945 (2006). https://doi.org/10.1038/nmeth940

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth940

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing