Mik Egbert G, Stap Jan, Sinaasappel Michiel, Beek Johan F, Aten Jacob A, van Leeuwen Ton G, Ince Can
Department of Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
Nat Methods. 2006 Nov;3(11):939-45. doi: 10.1038/nmeth940.
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 PO(2) 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.
分子氧是生物系统中的主要氧化剂。体内氧气的最终归宿是线粒体，在那里它被用于氧化磷酸化过程。该过程产生足以维持生命的高能磷酸盐的能力高度依赖于可用的氧气量。尽管有大量测量氧气的技术，但由于缺乏原位测量线粒体氧气的定量技术，主要的（病理）生理问题仍未得到解答。在此，我们证明通过利用内源性原卟啉IX（PpIX）的延迟荧光，可以在活细胞中直接测量线粒体PO₂，从而提供一种具有广泛应用潜力的技术。我们将该技术应用于不同的细胞系（V - 79中国仓鼠肺成纤维细胞、HeLa细胞和IMR 32 - K1神经母细胞瘤细胞），并首次直接测量了线粒体与细胞外液之间的氧梯度。
