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Structured illumination-based super-resolution live-cell quantitative FRET imaging
Photonics Research ( IF 7.6 ) Pub Date : 2023-05-01 , DOI: 10.1364/prj.485521
Zewei Luo, Ge Wu, Mengting Kong, Zhi Chen, Zhengfei Zhuang, Junchao Fan, and Tongsheng Chen

Förster resonance energy transfer (FRET) microscopy provides unique insight into the functionality of biological systems via imaging the spatiotemporal interactions and functional state of proteins. Distinguishing FRET signals from sub-diffraction regions requires super-resolution (SR) FRET imaging, yet is challenging to achieve from living cells. Here, we present an SR FRET method named SIM-FRET that combines SR structured illumination microscopy (SIM) imaging and acceptor sensitized emission FRET imaging for live-cell quantitative SR FRET imaging. Leveraging the robust co-localization prior of donor and accepter during FRET, we devised a mask filtering approach to mitigate the impact of SIM reconstruction artifacts on quantitative FRET analysis. Compared to wide-field FRET imaging, SIM-FRET provides nearly twofold spatial resolution enhancement of FRET imaging at sub-second timescales and maintains the advantages of quantitative FRET analysis in vivo. We validate the resolution enhancement and quantitative analysis fidelity of SIM-FRET signals in both simulated FRET models and live-cell FRET-standard construct samples. Our method reveals the intricate structure of FRET signals, which are commonly distorted in conventional wide-field FRET imaging.

中文翻译:

基于结构化照明的超分辨率活细胞定量 FRET 成像

Förster 共振能量转移 (FRET) 显微镜通过对蛋白质的时空相互作用和功能状态进行成像,提供了对生物系统功能的独特见解。区分来自亚衍射区域的 FRET 信号需要超分辨率 (SR) FRET 成像,但很难从活细胞中实现。在这里,我们提出了一种名为 SIM-FRET 的 SR FRET 方法,它结合了 SR 结构照明显微镜 (SIM) 成像和受体敏化发射 FRET 成像,用于活细胞定量 SR FRET 成像。在 FRET 期间利用供体和受体之前的稳健共定位,我们设计了一种掩模过滤方法来减轻 SIM 重建伪影对定量 FRET 分析的影响。与宽视野 FRET 成像相比,体内。我们在模拟 FRET 模型和活细胞 FRET 标准构建样本中验证了 SIM-FRET 信号的分辨率增强和定量分析保真度。我们的方法揭示了 FRET 信号的复杂结构,这些信号在传统的宽视场 FRET 成像中通常会失真。
更新日期:2023-05-02
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