Confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo.
Confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo.
复制标题
单线粒体超氧化物的共聚焦成像在完整的心脏或体内闪烁。
DOI：
10.3791/50818
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发表时间：
2013-11-05
期刊：
影响因子：
--
通讯作者：
Wang W
中科院分区：
文献类型：
--
作者：
Gong G;Wang W
线粒体是真核生物系统中重要的细胞器，负责能量产生和细胞内信号转导。线粒体功能障碍常常伴随并促成人类疾病。已经开发的用于评估线粒体功能和功能障碍的大多数方法是基于体外或离体测量。这些实验的结果在体内确定线粒体功能的能力有限。在这里，我们描述了一种新的方法，利用共聚焦扫描显微镜成像的完整组织在活的aminals，它允许在体内以实时的方式评价单个线粒体功能。首先，我们产生转基因小鼠表达线粒体靶向超氧化物指示剂，循环排列黄色荧光蛋白（mt-cpYFP）。将麻醉的mt-cpYFP小鼠固定在定制的舞台适配器上，并从后肢的暴露骨骼肌拍摄延时图像。随后处死小鼠，并将心脏设置为在37 °C下用生理溶液进行Langendorff灌注。将灌注的心脏定位在共聚焦显微镜载物台上的特殊腔室中，并且施加温和的压力以使心脏稳定并抑制心跳诱导的运动伪影。超氧化物闪光通过实时2D共焦成像以每秒一帧的频率检测。灌注溶液可以被修改以包含不同的呼吸底物或其他荧光指示剂。还可以调节灌注以产生疾病模型，例如缺血和再灌注。该技术是一种独特的方法，用于确定在完整组织和体内的功能的单一的神经元。
Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.
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影响因子：
3.6
作者：
Pieczenik, Steve R.;Neustadt, John
通讯作者：
Neustadt, John
单线粒体中的超氧化物闪光
影响因子：
64.5
作者：
Wang, Wang;Fang, Huaqiang;Groom, Linda;Cheng, Aiwu;Zhang, Wanrui;Liu, Jie;Wang, Xianhua;Li, Kaitao;Han, Peidong;Zheng, Ming;Yin, Jinhu;Wang, Weidong;Mattson, Mark P.;Kao, Joseph P. Y.;Lakatta, Edward G.;Sheu, Shey-Shing;Ouyang, Kunfu;Chen, Ju;Dirksen, Robert T.;Cheng, Heping
通讯作者：
Cheng, Heping
DOI：
10.1152/ajpheart.01345.2007
发表时间：
2008-05-01
影响因子：
4.8
作者：
Pasdois, Philippe;Beauvoit, Bertrand;Dos Santos, Pierre
通讯作者：
Dos Santos, Pierre
影响因子：
3
作者：
Lemieux, H.;Hoppel, C. L.
通讯作者：
Hoppel, C. L.
影响因子：
4.8
作者：
Hanson, GT;Aggeler, R;Remington, SJ
通讯作者：
Remington, SJ