Imaging Mitochondrial Calcium Signalling with Fluorescent Probes and Single or Two Photon Confocal Microscopy
Imaging Mitochondrial Calcium Signalling with Fluorescent Probes and Single or Two Photon Confocal Microscopy
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DOI：
10.1007/978-1-61779-382-0_14
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Duchen, Michael R.
中科院分区：
文献类型：
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作者：
Davidson, Sean M.;Duchen, Michael R.
线粒体中钙离子的浓度对线粒体功能有深远的影响，在生理浓度下调节呼吸活动，同时在钙超载时造成致命的损伤。“rhod”系列钙敏感荧光染料倾向于在线粒体中优先积累，尽管线粒体钙测量的可靠性主要取决于线粒体内染料的分配，这可能因制剂而异。本文描述了使用单光子或双光子共聚焦显微镜并将成像与另一种细胞质钙传感染料相结合的方法来帮助验证和定量线粒体钙浓度。线性解混的方法，以分离荧光信号基于不同的激发或发射光谱被概述，并为说明的目的，适用于从线粒体和核仁内染料起源的rhod-2信号的分离。
The concentration of calcium ions in the mitochondria has a profound impact on mitochondrial function, modulating respiratory activity at physiological concentrations, while causing lethal damage during calcium overload. The "rhod" series of calcium sensitive fluorescent dyes tend to accumulate preferentially in mitochondria, although the reliability of mitochondrial calcium measurements depends critically on the partitioning of dye within the mitochondria which can vary between preparations. Methods are described to aid verification and quantification of the mitochondrial calcium concentration using single or two photon confocal microscopy and combining the imaging with another cytosolic calcium sensing dye. The method of linear unmixing to separate fluorescent signals based on either differing excitation or emission spectra is outlined and for the purposes of illustration is applied to the separation of rhod-2 signals originating from dye within the mitochondrial and nucleoli.