Optical section structured illumination-based Förster resonance energy transfer imaging

Abstract

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were $E_{C32V}^{\mathrm{OSF}}=0.32\pm 0.02,E_{C17V}^{\mathrm{OSF}}=0.38\pm 0.02$ , and $E_{C5V}^{\mathrm{OSF}}=0.45\pm 0.03$ , and the measured acceptor-to-donor concentration ratios ( $R_c$ ) were $R_{C32V}^{\mathrm{OSF}}=1.07\pm 0.03$ , $R_{C17V}^{\mathrm{OSF}}=1.09\pm 0.03$ , and $R_{C5V}^{\mathrm{OSF}}=1.02\pm 0.04$ , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

Keywords: Förster resonance energy transfer; living cells; optical section; quantitative measurement; structured illumination microscopy.