A prism-type total internal reflection fluorescence microscope was used to acquire fluorescence emission signals from Cy3 and Cy5 by a water immersion objective lens (UPlanApo 60×, Olympus). The laser scattering was rejected through a 550-nm long-pass filter. The fluorescence emission light was further separated into donor and acceptor signals with a 630- or 635-nm dichroic mirror (Chroma) and projected onto a back-illuminated electron-multiplying charge-coupled device (Andor) with a time resolution of 30 to 100 ms. Fluorescence signals of the donor and acceptor were amplified by a gain before camera readout. Thus, both recorded fluorescence intensities of Cy3 and Cy5 are in an arbitrary unit (a.u.), proportional to their photon counts. FRET efficiency is determined by the ratio of intensities, Intensityacceptor/(Intensitydonor + Intensityacceptor) after correcting for cross-talk between the donor and acceptor channels. All data were analyzed by MATLAB codes and plotted in Origin.

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