Structured illumination-based super-resolution Förster resonance energy transfer microscopy (SIM-FRET) provides an approach to resolving molecular behavior localized in intricate biological structures in living cells. However, SIM reconstruction artifacts will decrease the quantitative analysis fidelity of SIM-FRET signals. To address these issues, we have developed a method called HiFi spectrum optimization SIM-FRET (HiFi-SO-SIM-FRET), which uses optimized Wiener parameters in the two-step spectrum optimization to suppress sidelobe artifacts and achieve super-resolution quantitative SIM-FRET. We validated our method by demonstrating its ability to reduce reconstruction artifacts while maintaining the accuracy of FRET signals in both simulated FRET models and live-cell FRET-standard construct samples. In summary, HiFi-SO-SIM-FRET provides a promising solution for achieving high spatial resolution and reducing SIM reconstruction artifacts in quantitative FRET imaging.

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.

由于具有低光毒性、高速宽视场以及多通道三维超分辨成像能力,超分辨结构照明显微术(SR-SIM)特别适合用于活细胞中动态精细结构的实时检测研究。超分辨结构照明显微图像重建算法(SIM-RA)对SR-SIM的成像质量具有决定性影响。本文首先简要介绍了超分辨显微术的发展现状,阐述了研究SR-SIM图像重建算法的必要性;然后介绍了SR-SIM的成像原理,并重点介绍了SR-SIM图像重建算法,包括SR-SIM中频繁使用的去卷积重建算法、SR-SIM校准与重建过程中参数值获取的算法,以及目前发展的超分辨结构照明显微图像重建算法,并介绍了SR-SIM工具箱;最后总结了当前发展超分辨结构照明显微图像重建算法需解决的5个问题。