IIem-spFRET: improved Iem-spFRET method for robust FRET measurement.

We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence.

Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells.

3-cube-based Förster resonance energy transfer (FRET) microscopy, a sensitized acceptor FRET quantification method, has been widely used to visualize dynamic protein-protein interaction in living cells. Determining the FRET sensitized-quenching transition factor (G factor) of a particular donor-acceptor pair and optical system is crucial for 3-cube FRET quantification. We here improved the acceptor photobleaching-based G factor determination method (termed as mPb-G) and the two-plasmid-based G factor determination method (termed as mTP-G) for rapid and reliable measurement of the G factor.

Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells.

With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E ) and acceptor-to-donor concentration ratio (R C ) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods.

Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway.

Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells.

Spectral measurement of fluorescence resonance energy transfer (FRET), spFRET, is a widely used FRET quantification method in living cells today. We set up a spectrometer-microscope platform that consists of a miniature fiber optic spectrometer and a widefield fluorescence microscope for the spectral measurement of absolute FRET efficiency (E) and acceptor-to-donor concentration ratio (R(C)) in single living cells. The microscope was used for guiding cells and the spectra were simultaneously detected by the miniature fiber optic spectrometer.

Spectral measurement of acceptor-to-donor extinction coefficient ratio in living cells.

This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio (γ(λex)) of acceptor-to-donor in living cells at excitation wavelength λex, which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ(λex) by spectrally unmixing the emission spectrum of a donor-acceptor tandem construct under λex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ(458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method.