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 cel...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 SIMFRET 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.展开更多
Forster 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 s...Forster 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.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.62135003)Key-Area Research and Development Program of Guangdong Province(Grant No.2022B0303040003).
文摘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 SIMFRET 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.
基金National Natural Science Foundation of China(62135003,62103071)Key-Area Research and Development Program of Guangdong Province(2022B0303040003)+2 种基金Natural Science Foundation of Chongqing(cstc2021jcyj-msxm X0526,sl202100000288)Science and Technology Program of GuangzhouScience and Technology Research Program of Chongqing Municipal Education Commission(KJQN202100630)。
文摘Forster 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.
