Affiliation:
1. SCNU Qingyuan Institute of Science and Technology Innovation Co., Ltd.
Abstract
Reliable measurements of calibration parameters are crucial for quantitative three-cube Förster resonance energy transfer (FRET) measurements. Here we have developed a single-cell-based calibration method (SCC-FRET), which can simultaneously obtain spectral crosstalk correction parameters (β and δ) and calibration parameters (G and k) of a quantitative FRET system by imaging a cell expressing one kind of standard FRET plasmid with a known FRET efficiency (E) and the donor-to-acceptor concentration ratio (RC). We performed the SCC-FRET method on a three-cube FRET microscopy for the cells expressing C5V, and obtained β = 0.150 ± 0.000, δ = 0.610 ± 0.000, G = 2.840 ± 0.065, and k = 0.847 ± 0.013. These parameters were used to measure the E and RC values of C17V and C32V constructs in living cells and obtained EC17V = 0.382 ± 0.010 and EC32V = 0.311 ± 0.007, RC17V = 1.010 ± 0.023 and RC32V = 1.050 ± 0.022, consistent with the reported values, demonstrating the effectiveness of the the SCC-FRET method. We also performed the SCC-FRET method for the cells with different S/N levels (S/N > 10, 10 > S/N > 3, 3 > S/N > 1, respectively), and obtained consistent system calibration parameters under different S/N levels, indicating excellent robustness. SCC-FRET requires only imaging a cell expressing one kind of standard FRET plasmid for measuring all calibration parameters under identical imaging conditions, rendering the SCC-FRET method extremely convenient, accurate, and robust. The SCC-FRET provides strong support for expanding the biological application of quantitative FRET analysis in living cells.
Funder
National Natural Science Foundation of China
Science and Technology Program of Guangzhou
Subject
Atomic and Molecular Physics, and Optics
Cited by
1 articles.
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