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.

Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.

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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.

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