No 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 EnormalC32normalVOSF=0.32±0.5em0.02,0.5emEnormalC17normalVOSF=0.38±0.02, and EnormalC5normalVOSF=0.45±0.03, and the measured acceptor‐to‐donor concentration ratios (Rc) were RnormalC32normalVOSF=1.07±0.03, RnormalC17normalVOSF=1.09±0.03, and RnormalC5normalVOSF=1.02±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.

Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRETsensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: ( 1) predetermining the ratio ranges of the sensitized emission of acceptor (F C ) to the donor excitation and donor channel image (I DD [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their F C /I DD (DA) values; (4) linearly fitting F C /I AA (DA) (acceptor excitation and acceptor channel image) to I DD (DA)/I AA (DA)for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.

Accurate predetermination of the quantum yield ratio (QA/QD) and the extinction coefficient ratio (KA/KD) between acceptor and donor is a prerequisite for quantitative fluorescence resonance energy transfer (FRET) imaging. We here propose a method to measure KA/KD and QA/QD by measuring the excitation–emission spectra (ExEm-spectra) of one dish of cells expressing m (≥3) kinds of FRET constructs. The ExEm-spectra images are unmixed to obtain the weight maps of donor (WD), acceptor (WA), and acceptor sensitization (WS). For each cell, the frequency distribution plots of the WS/WD and WS/WA images are fitted by using a single-Gaussian function to obtain the peak values of WS/WD (SD) and WS/WA (SA). The statistical frequency-SD/SA plots from all cells are fitted by using a multi-Gaussian function to obtain the peak values of both SD and SA, and then the ranges of WS/WD (RSD) and WS/WA (RSA) for each FRET construct are predetermined. Based on the predetermined RSD and RSA values of FRET constructs, our method is capable of automatically classifying cells expressing different FRET constructs. Finally, the WS/WD–WA/WD plot from different kinds of cells is linearly fitted to obtain KA/KD and QA/QD values.