Dixit Ram, Cyr Richard
The Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA.
Plant J. 2003 Oct;36(2):280-90. doi: 10.1046/j.1365-313x.2003.01868.x.
The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.
绿色荧光蛋白（GFP）和其他固有荧光蛋白（IFP）是常用的报告分子，因为它们能够使活样本中的细胞成分可视化。IFP技术使观察活细胞中的动态过程成为可能，但使用荧光显微镜（宽视野和共聚焦）进行长时间观察会导致大量光能暴露。因此，细胞有可能受到光诱导的损伤，从而改变细胞生理状态并混淆观察结果。为了了解长时间观察对细胞的影响，我们获得了关于光能剂量和观察条件对烟草BY-2细胞生理影响的定量信息。我们的结果表明，激发光强度与有丝分裂停滞之间存在非线性关系，有丝分裂停滞的频率取决于吸收激发光的IFP的存在。此外，荧光显微镜会诱导活性氧（ROS）的产生，这是通过加载氧化敏感染料的BY-2细胞检测到的，如果细胞表达吸收激发光能的IFP，ROS的产生水平会增加。染料氧化遵循S形动力学，如果细胞暴露于低辐照水平，氧化是可逆的。此外，染料氧化速率与激发光强度呈非线性关系，光漂白、有丝分裂停滞和染料氧化之间存在良好的相关性。这些数据突出了ROS清除对于正常有丝分裂进程的重要性，并为明智地选择避免导致ROS积累和生理损伤的光漂白的条件提供了参考。
