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Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX

Nature Methods volume 3, pages 939945 (2006) | Download Citation



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.

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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


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

    • Egbert G Mik
    •  & Can Ince
  2. Department of Anesthesiology, Erasmus Medical Center, University of Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

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

    • Jan Stap
    •  & Jacob A Aten
  4. Laser Center, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.

    • Michiel Sinaasappel
    • , Johan F Beek
    •  & Ton G van Leeuwen
  5. Biophysical Engineering, Biomedical Technology Institute, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands.

    • Ton G van Leeuwen


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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.

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.

Corresponding author

Correspondence to Egbert G Mik.

Supplementary information

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  1. 1.

    Supplementary Fig. 1

    Extended calibration curve in HeLa cells.

  2. 2.

    Supplementary Fig. 2

    Simultaneous delayed fluorescence and phosphorescence lifetime measurements in a homogenous solution.

  3. 3.

    Supplementary Methods

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