Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai 200241, China.
J Am Chem Soc. 2020 Apr 22;142(16):7532-7541. doi: 10.1021/jacs.0c00771. Epub 2020 Apr 13.
Mitochondrial oxidative stress and energy metabolism are vital biological events and are involved in various physiological and pathological processes such as apoptosis and necrosis. However, it remains unclear how the dynamic patterns of mitochondrial hydrogen peroxide (HO) and adenosine-5'-triphosphate (ATP) change in these events and, more importantly, how they affect each other. Herein, we developed a single two-photon fluorescence-lifetime-based probe (TFP), which offered real-time imaging and the simultaneous determination of mitochondrial HO and ATP changes in two well-separated fluorescence channels without spectral crosstalk. The fluorescence lifetime of TFP exhibited good responses and selectivity in the detection ranges of 0.4-10 μM HO and 0.5-15 mM ATP, taking advantage of accuracy and the quantitative ability of fluorescence lifetime imaging. Using this useful probe, we studied the relationship between HO and ATP in mitochondria and visualized the dynamic level changes of mitochondrial HO and ATP induced by the superoxide anion (O). It was discovered that O stimulation in a short period of time (8 min) temporarily changes the levels of HO and ATP in mitochondria, and neurons were capable of recovering to the initial state in a short time. However, increasing time of up to 50 min of O stimulation led to permanent oxidative damage and an energy deficiency. Meanwhile, it was first found that the exogenous stimulation of O and HO had different impacts on the levels of mitochondrial HO and ATP, in which O demonstrated more severe and negative consequences. As a matter of fact, this work not only has provided a general molecular design methodology for multiple species imaging but also has revealed oxidative-stress-induced intracellular functions related to HO and ATP in mitochondria based on this developed TFP probe.
线粒体氧化应激和能量代谢是至关重要的生物学事件，涉及到细胞凋亡和坏死等多种生理和病理过程。然而，目前尚不清楚线粒体过氧化氢（HO）和三磷酸腺苷（ATP）的动态模式在这些事件中是如何变化的，更重要的是，它们是如何相互影响的。在此，我们开发了一种单双光子荧光寿命基探针（TFP），它可以在两个完全分离的荧光通道中实时成像并同时测定线粒体 HO 和 ATP 的变化，而不会产生光谱串扰。TFP 的荧光寿命在 0.4-10 μM HO 和 0.5-15 mM ATP 的检测范围内表现出良好的响应和选择性，利用了荧光寿命成像的准确性和定量能力。利用这种有用的探针，我们研究了线粒体中 HO 和 ATP 之间的关系，并可视化了超氧阴离子（O）诱导的线粒体 HO 和 ATP 的动态水平变化。结果发现，O 在短时间（8 分钟）内刺激会暂时改变线粒体中 HO 和 ATP 的水平，神经元能够在短时间内恢复到初始状态。然而，O 刺激时间增加到 50 分钟，会导致永久性氧化损伤和能量缺乏。同时，首次发现 O 和 HO 的外源性刺激对线粒体 HO 和 ATP 的水平有不同的影响，其中 O 表现出更严重和负面的后果。事实上，这项工作不仅为多种物种成像提供了一种通用的分子设计方法，而且还基于这种开发的 TFP 探针揭示了与线粒体中 HO 和 ATP 相关的氧化应激诱导的细胞内功能。
