Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.
Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.
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DOI：
10.3389/fphys.2023.1257739
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发表时间：
2023
影响因子：
4
通讯作者：
中科院分区：
文献类型：
--
作者：
前言：MitoView 633是一种远红荧光染料，如能斯特方程所描述的那样，它能够以膜电位依赖的方式在线粒体内蓄积。这一特性使其成为生物能量学研究的一个有前途的候选者，特别是作为线粒体膜电位(DYM)的一个强有力的指示器。尽管它有很大的潜力，但它在活细胞成像中的用途还没有得到很好的表征。方法：本研究旨在确定MitoView 633在活细胞中的光谱特性，并评估其线粒体染色、光漂白和DYM去极化过程中的动力学。用共聚焦显微镜观察了MitoView 633与其他荧光载体如MitoSOX Red和Fluo-4 AM在心肌细胞中的共染色和成像。结果和讨论：光谱分析显示MitoView 633的发射波长为660±50 nm，与常用的DYM指示剂四甲基罗丹明甲酯(TMRM)相比，MitoView 633的热稳定性更好，后者的发射波长为605±25 nm。共聚焦成像清楚地显示了MitoView 633‘S在线粒体基质中的特定定位，并与公认的线粒体标记MitoTracker Green进行了共定位。此外，我们的调查显示MitoView 633在推荐的体外浓度下表现出最小的光漂白。此外，在线粒体解偶联剂乙酰氰化物-对三氟甲氧基苯肼(FCCP)诱导的DYM去极化过程中，MitoView 633的荧光动力学与TMRM的动力学相似。重要的是，MitoView 633显示出与MitoSOX Red和Fluo-4 AM共染色的兼容性，能够同时监测完整细胞中的DYM、线粒体ROS和胞浆钙离子。结论：这些发现共同强调MitoView 633是一种在活细胞成像应用中单独或联合评估DYM和其他指标的极佳分子探针。
Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633’s specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.
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影响因子：
3.7
作者：
Zhao Z;Gordan R;Wen H;Fefelova N;Zang WJ;Xie LH
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Xie LH
DOI：
10.1007/s00424-021-02524-3
发表时间：
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作者：
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通讯作者：
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