复原单分子荧光共振能量转移(smFRET)研究中复原FRET真实效率需要减弱三重态的影响，这一成果由美国圣裘德儿童研究医院Scott C. Blanchard研究组经过不懈努力而取得。相关论文于2024年6月14日发表于国际学术期刊《自然-方法学》杂志。

据介绍，用于量化生物大分子内部成分随时间变化和构象变化的smFRET需要较高的照明强度，才能从单个荧光团中恢复强大的光子发射流。

研究人员展示了在弱激发极限外，以及荧光集团必须经历多次快速激发和灭活循环的情况下，非荧光、激发诱导的三重态（其寿命比光子发射单态长几个数量级）会降低供体和受体荧光团的光子发射流，从而导致FRET效率随光照强度变化而变化。

这些变化通常未被考虑在内，因此，需要抑制激发态累积的稳健策略，以恢复准确、精确的FRET效率，进而估算距离。该研究提出了稳健的三重态抑制和数据校正策略，使恢复的FRET效率更接近真实值，从而扩展了smFRET的空间和时间分辨率。

附：英文原文

Title: Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation

Author: Pati, Avik K., Kilic, Zeliha, Martin, Maxwell I., Terry, Daniel S., Borgia, Alessandro, Bar, Sukanta, Jockusch, Steffen, Kiselev, Roman, Altman, Roger B., Blanchard, Scott C.

Issue&Volume: 2024-06-14

Abstract: Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.

DOI: 10.1038/s41592-024-02293-8

Source: https://www.nature.com/articles/s41592-024-02293-8