Optimizing Methods to Recover Absolute FRET Efficiency from Immobilized Single Molecules
Optimizing Methods to Recover Absolute FRET Efficiency from Immobilized Single Molecules
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DOI：
10.1016/j.bpj.2010.04.063
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发表时间：
2010-08-04
影响因子：
3.4
通讯作者：
Bowen, Mark E.
中科院分区：
文献类型：
--
作者：
McCann, James J.;Choi, Ucheor B.;Bowen, Mark E.
基于显微镜的荧光共振能量转移（FRET）实验测量供体和受体的强度，通过隔离这些信号与一系列光学元件。因为这种滤波丢弃了部分光谱，所以观察到的FRET效率取决于所使用的滤波器集。同样，观察到的FRET效率也受到荧光团量子产率差异的影响。恢复绝对FRET效率需要对这些影响进行标准化，以考虑供体和受体荧光团之间的量子产率和检测效率的差异。如果没有这种校正，只有在光物理和仪器性能保持不变的情况下，FRET在多个实验中是一致的。在这里，我们提出了什么是，据我们所知，第一个系统的研究方法，以恢复真正的FRET效率使用DNA尺子与已知的荧光团分离。我们改变光学元件，有目的地改变观察到的FRET和检测蛋白质样品，以获得不同于DNA样品的量子产率。计算仪器传输的校正减少了FRET偏差，这可以方便比较不同仪器的结果。经验归一化更有效，但需要大量的努力。基于单分子光漂白的标准化是最有效的，这取决于它是如何应用的。令人惊讶的是，单分子伽玛归一化降低了DNA FRET分布的峰宽，因为异常的伽玛值对应于FRET异常值。因此，分子间γ的变化对FRET分布具有不可识别的影响，必须考虑从分布宽度中提取样品动力学信息。
Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.