Ward Manus W
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.
Methods Mol Biol. 2010;591:335-51. doi: 10.1007/978-1-60761-404-3_20.
The changes that occur in electrochemical gradients across biological membranes provide us with invaluable information on physiological responses, pathophysiological processes and drug actions/toxicity. This chapter aims to provide researchers with sufficient information to carry out a quantitative assessment of mitochondrial energetics at a single-cell level thereby providing output on changes in the mitochondrial membrane potential (Deltapsi(m)) through the utilization of potentiometric fluorescent probes (TMRM, TMRE, Rhodamine 123). As these cationic probes behave in a Nernstian fashion, changes at the plasma membrane potential (Deltapsi(p)) need also to be accounted for in order to validate the responses obtained with Deltapsi(m)-sensitive fluorescent probes. To this end techniques that utilize Deltapsi(p)-sensitive anionic fluorescent probes to monitor changes in the plasma membrane potential will also be discussed. In many biological systems multiple changes occur at both a Deltapsi(m) and Deltapsi(p) level that often makes the interpretation of the cationic fluorescent responses much more difficult. This problem has driven the development of computational modelling techniques that utilize the redistribution properties of the cationic and anionic fluorescent probes within the cell to provide output on changes in Deltapsi(m) and Deltapsi(p).
跨生物膜的电化学梯度变化为我们提供了有关生理反应、病理生理过程以及药物作用/毒性的宝贵信息。本章旨在为研究人员提供足够的信息，以便在单细胞水平上对线粒体能量学进行定量评估，从而通过使用电位荧光探针（四甲基罗丹明甲酯、四甲基罗丹明乙酯、罗丹明123）来输出线粒体膜电位（Δψm）的变化。由于这些阳离子探针以能斯特方式起作用，为了验证用对Δψm敏感的荧光探针获得的反应，还需要考虑质膜电位（Δψp）的变化。为此，还将讨论利用对Δψp敏感的阴离子荧光探针来监测质膜电位变化的技术。在许多生物系统中，Δψm和Δψp水平都会发生多种变化，这常常使阳离子荧光反应的解释变得更加困难。这个问题推动了计算建模技术的发展，该技术利用细胞内阳离子和阴离子荧光探针的重新分布特性来输出Δψm和Δψp的变化。
