Fig. 1. Flow chart and basic principle of HiFi-SO-SIM-FRET. (a) The HiFi-SO-SIM-FRET flow chart includes obtaining three-channel FRET structured illumination modulated raw image stacks, SR-SIM image reconstruction of the three-channel FRET imaging based on spectrum optimization, background subtraction, binary mask filtering, and quantitative sensitized emission FRET measurement. (b) Composite filter for acceptor (AA) and donor (DD) channels of Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM. (c) Composite filter ratio of acceptor and donor channels for Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM. (d) Intensity profiles of the red lines in panel (b) and intensity profiles of the blue lines in panel (c).

Fig. 2. Feasibility of HiFi-SO-SIM for the elimination of sidelobe artifacts and quantitative calculation of FRET. (a) GT of simulated rings, WF imaging, and corresponding SR images reconstructed by Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM. (b) Magnified images of the GT, WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM results corresponding to the yellow-boxed region in panel (a). (c) Magnified images of the GT, WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM results corresponding to the red-boxed region in panel (e). (d) Intensity profiles of the red lines in panel (b). Red arrows indicate sidelobe artifacts produced by OTF mismatch. (e) FRET ED images of GT, WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM. (f) ED profiles of the red lines in panel (c). Red arrows indicate the erroneous FRET signals generated by sidelobe artifacts. Scale bar: 50 pixels.

Fig. 3. Performance of HiFi-SO-SIM in FRET star-like pattern simulation. (a) Three-channel images of simulation FRET models of GT, WF, HiFi-SIM, and HiFi-SO-SIM. (b) FRET ED images of GT, WF, HiFi-SIM, and HiFi-SO-SIM. (c) FRET RC images of GT, WF, HiFi-SIM, and HiFi-SO-SIM. (d) Intensity profiles of the white solid lines in panel (a). (e) Corresponding histograms of ED in panel (b). (f) Corresponding histograms of RC in panel (c). Scale bar: 100 pixels.

Fig. 4. Performance of HiFi-SO-SIM for quantitative FRET measurements in live cells. (a) Three-channel intensity WF images of ActA-G17M. (b) WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM magnified images of the green box area in the corresponding panel (a). (c) Intensity profiles of the green line in panel (b). (d) Pseudo-color images of WF ED using raw three-channel images. (e) WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM magnified images of the red box area in the corresponding panel (d). (f) Pseudo-color images of WF RC using raw three-channel images. (g) WF, Wiener-SIM, HiFi-SIM, and HiFi-SO-SIM magnified images of the yellow box area in the corresponding panel (f). (h) ED profiles of the red lines in panel (e). Scale bar: 5μm.

Fig. 5. Comparison of statistical ED and RC values using different methods. (a) Statistical ED values using different methods. (b) Statistical RC values using different methods. The unpaired student’s t-test is used for data [(a) and (b)]. ∗∗, p<0.01; ∗∗∗, p<0.001; ns, p>0.05.