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Optical microwell array for large scale studies of single mitochondria metabolic responses

机译:光学微孔阵列用于单线粒体代谢反应的大规模研究

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Microsystems based on microwell arrays have been widely used for studies on single living cells. In this work, we focused on the subcellular level in order to monitor biological responses directly on individual organelles. Consequently, we developed microwell arrays for the entrapment and fluorescence microscopy of single isolated organelles, mitochondria herein. Highly dense arrays of 3-μm mean diameter wells were obtained by wet chemical etching of optical fiber bundles. Favorable conditions for the stable entrapment of individual mitochondria within a majority of microwells were found. Owing to NADH auto-fluorescence, the metabolic status of each mitochondrion was analyzed at resting state (Stage 1), then following the addition of a respiratory substrate (Stage 2), ethanol herein, and of a respiratory inhibitor (Stage 3), antimycin A. Mean levels of mitochondrial NADH were increased by 29 % and 35 % under Stages 2 and 3, respectively. We showed that mitochondrial ability to generate higher levels of NADH (i.e., its metabolic performance) is not correlated either to the initial energetic state or to the respective size of each mitochondrion. This study demonstrates that microwell arrays allow metabolic studies on populations of isolated mitochondria with a single organelle resolution.
机译:基于微孔阵列的微系统已被广泛用于单个活细胞的研究。在这项工作中,我们专注于亚细胞水平,以便直接监测单个细胞器上的生物学反应。因此,我们开发了微孔阵列,用于单个分离的细胞器,线粒体的捕获和荧光显微镜检查。通过对光纤束进行湿化学蚀刻,获得了平均孔径为3μm的高密度阵列。发现在大多数微孔中稳定捕获单​​个线粒体的有利条件。由于NADH自发荧光,在静息状态(阶段1),然后在此添加呼吸底物(阶段2),乙醇和呼吸抑制剂(阶段3),抗霉素之后,分析了每个线粒体的代谢状态。答:在阶段2和阶段3中,线粒体NADH的平均水平分别增加了29%和35%。我们表明,线粒体产生更高水平的NADH的能力(即其代谢性能)与初始能量状态或每个线粒体的各自大小均无关。这项研究表明,微孔阵列可以单细胞器分辨率对孤立的线粒体群体进行代谢研究。

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