Measuring calibration factors by imaging a dish of cells expressing different tandem constructs plasmids

Abstract

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.

Optical section structured illumination‐based Förster resonance energy transfer imaging

“…The detailed and values are shown in Figure 7. The and values measured by the OS‐SIM‐FRET method are consistent with the reported in literature [32, 36] indicating that OS‐SIM is compatible with E‐FRET method.…”

“…And the peak value of the G distribution measured under OS-SIM mode is smaller than the peak value measured under WF mode (Figure 5). The The E D and R C values measured by the OS-SIM-FRET method are consistent with the reported in literature [32,36] indicating that OS-SIM is compatible with E-FRET method.…”

“…The four spectral crosstalk coefficients measured by using donor‐only specimen and acceptor‐only specimen respectively and the calibration factors G and k measured by using Hela cells expressing Cerulean‐5‐Venus under OS‐SIM imaging mode were consistent with those measured under conventional widefield imaging mode. Further, the measured FRET efficiency () and concentration ratio () values of standard FRET plasmids under OS‐SIM mode are consistent with the reported values [30, 32]. Our data firmly demonstrate that E‐FRET can be integrated with OS‐SIM as an OS‐SIM‐FRET microscope with high resolution.…”

Measuring System Calibration Factors by Unmixing the Excitation–Emission Spectra of One Dish of Cells

“…CTV, C32V, C17V, and C5V were transfected, respectively, into the four wells of cells for 4-6 h by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells in the four wells were then detached and subsequently mixed into a 35-mm glass dish and cultured for 12-24 h in CO 2 incubators (Yin et al, 2021).…”