Fercher Andreas, O'Riordan Tomas C, Zhdanov Alexander V, Dmitriev Ruslan I, Papkovsky Dmitri B
Biochemistry Department, University College Cork, Cork, Ireland.
Methods Mol Biol. 2010;591:257-73. doi: 10.1007/978-1-60761-404-3_16.
Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O(2) concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.
许多反映细胞线粒体功能和代谢状态的参数，包括线粒体膜电位、活性氧、三磷酸腺苷（ATP）、烟酰胺腺嘌呤二核苷酸（NADH）、离子梯度和离子通量（钙离子、氢离子），都适合通过活细胞成像进行分析，并且在许多实验室中被广泛应用。然而，一种关键的代谢物——细胞内氧气——目前尚未得到常规分析。在此，我们展示了几种成像技术，这些技术使用细胞内加载的磷光氧敏感探针，能够实时监测活的呼吸细胞中的氧气以及细胞对刺激的代谢反应。这些技术包括用于监测细胞呼吸相对变化的传统宽场荧光显微镜、提供细胞内/细胞附近氧气浓度定量读数的微秒荧光寿命成像（FLIM）格式，以及用于监测单个悬浮细胞代谢反应的活细胞阵列装置。文中给出了这些应用中每个典型实验的逐步操作步骤以及故障排除指南。
