High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy
High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy
复制标题
DOI：
10.1529/biophysj.104.055442
复制
发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Piston, DW
中科院分区：
文献类型：
--
作者：
Rizzo, MA;Piston, DW
荧光蛋白标记靶分子之间的共振能量转移（FRET）检测是一种有价值的策略，用于测量蛋白质-蛋白质相互作用和其他细胞内过程。尽管FRET的效用，广泛应用于生物学问题和高通量筛选这种技术已受到限制的低对比度的测量策略，依赖于检测敏化发射或光破坏的样品。在这里，我们报告的FRET检测策略的基础上检测去极化敏化发射。在没有FRET的情况下，我们表明，从供体荧光蛋白的荧光发射是高度偏振的。只有在能量转移的存在下才能观察到荧光发射的去偏振。一个简单的检测策略，适用于荧光显微镜使用激光扫描和宽视场的方法。这种方法能够区分连接和未连接的Cerulean和Venus荧光蛋白在活细胞中的FRET具有比其他方法更大的动态范围。
Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.