In vivo oxygen imaging using green fluorescent protein
In vivo oxygen imaging using green fluorescent protein
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DOI：
10.1152/ajpcell.00067.2006
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发表时间：
2006-10-01
影响因子：
5.5
通讯作者：
Sato, Michihiko
中科院分区：
文献类型：
--
作者：
Takahashi, Eiji;Takano, Tomohiro;Sato, Michihiko
体内氧气测量是了解生物系统如何动态适应氧气供应减少的关键。高空间分辨率氧成像特别重要，因为最近的研究解决了组织内和细胞内氧浓度异质性在健康和疾病中的重要性。在这里，我们报告了一种利用绿色荧光蛋白（GFP）对器官中的氧气进行体内分子成像的新技术。表达gfp的COS-7细胞在强蓝光下短暂光激活，同时将氧浓度从10%降低到< 0.001%。在< 2%氧的光激活下（GFP荧光的红移）出现红色荧光（激发520 ~ 550 nm，发射> ~ 580 nm）。细胞再氧化后红移消失，说明只要细胞处于缺氧状态，红移是稳定的。在从GFP敲入小鼠（绿色小鼠）心脏分离的单个心肌细胞中也显示了GFP荧光的红移。然后，我们尝试了器官缺氧的体内分子成像。在阻止大气中氧气扩散的条件下，利用宏观光学可以在绿色小鼠缺血的肝脏和肾脏中成像红移。在从绿色小鼠分离的晶体灌注的跳动心脏中，在冠状动脉结扎的远端心外膜上显示出明显的红移空间异质性。我们的结论是，目前的技术使用绿色荧光蛋白作为氧指示剂可能允许在体内的器官氧分子成像。
In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to < 0.001%. Red fluorescence (excitation 520-550 nm, emission > 580 nm) appeared after photoactivation at < 2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.