• Photonics Research
  • Vol. 11, Issue 5, 887 (2023)
Zewei Luo1、2、†, Ge Wu1、2、†, Mengting Kong1、2, Zhi Chen1、2, Zhengfei Zhuang1、2、3, Junchao Fan4、5、*, and Tongsheng Chen1、2、3、6、*
Author Affiliations
  • 1Key Laboratory of Laser Life Science, Ministry of Education, College of Biophotonics, South China Normal University, Guangzhou 510631, China
  • 2Guangdong Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
  • 3SCNU Qingyuan Institute of Science and Technology Innovation, South China Normal University, Qingyuan 511520, China
  • 4Chongqing Key Laboratory of Image Cognition, College of Computer Science and Technology, Chongqing University of Posts and Telecommunications, Chongqing 400065, China
  • 5e-mail: fanjc@cqupt.edu.cn
  • 6e-mail: chentsh@scnu.edu.cn
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    DOI: 10.1364/PRJ.485521 Cite this Article Set citation alerts
    Zewei Luo, Ge Wu, Mengting Kong, Zhi Chen, Zhengfei Zhuang, Junchao Fan, Tongsheng Chen. Structured illumination-based super-resolution live-cell quantitative FRET imaging[J]. Photonics Research, 2023, 11(5): 887 Copy Citation Text show less

    Abstract

    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.
    Dθ,nX(r)={S(r)·[1+mθX·cos(pθX·r+ϕθX+2πn3)]}HX(r),

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    [S˜θ,0X(k)S˜θ,1X(kpθX)S˜θ,1X(k+pθX)]=[1mθXei(ϕθX)/2mθXei(ϕθX)/21mθXei(ϕθX+2π3)/2mθXei(ϕθX+2π3)/21mθXei(ϕθX+4π3)/2mθXei(ϕθX+4π3)/2]1[D˜θ,0X(k)D˜θ,1X(k)D˜θ,1X(k)],

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    ISIMX(r)=ifft[θ,nS˜θ,nXH˜*X(k+npθX)θ,n|H˜X(k+npθX)|2+w2A˜(k)],

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    ED=ISIMDAa·(ISIMAAc·ISIMDD)d·(ISIMDDb·ISIMAA)ISIMDAa·(ISIMAAc·ISIMDD)d·(ISIMDDb·ISIMAA)+G·ISIMDD,

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    RC=K·ISIMAA[ISIMDAa·(ISIMAAc·ISIMDD)d·(ISIMDDb·ISIMAA)]/G+ISIMDD,

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    PCCmap(r)=[ISIMDD(r)IAVGDD]·[ISIMAA(r)IAVGAA]r[ISIMDD(r)IAVGDD]2·r[ISIMAA(r)IAVGAA]2,

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    MOCmap(r)=ISIMDD(r)·ISIMAA(r)r[ISIMDD(r)]2·r[ISIMAA(r)]2,

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    Bcolocalmask(r)={1,if PCCmap(r)·MOCmap(r)>th0,otherwise.

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    ED=ED·Bcolocalmask,

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    RC=RC·Bcolocalmask.

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    Zewei Luo, Ge Wu, Mengting Kong, Zhi Chen, Zhengfei Zhuang, Junchao Fan, Tongsheng Chen. Structured illumination-based super-resolution live-cell quantitative FRET imaging[J]. Photonics Research, 2023, 11(5): 887
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