摘要
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.
基金
National Natural Science Foundation of China(62135003,62103071)
Key-Area Research and Development Program of Guangdong Province(2022B0303040003)
Natural Science Foundation of Chongqing(cstc2021jcyj-msxm X0526,sl202100000288)
Science and Technology Program of Guangzhou
Science and Technology Research Program of Chongqing Municipal Education Commission(KJQN202100630)。