Im-SCC-FRET: improved single-cell-based calibration of a FRET system.

We recently developed a SCC-FRET (single-cell-based calibration of a FRET system) method to quantify spectral crosstalk correction parameters (ß and δ) and system calibration parameters (G and k) of a Förster resonance energy transfer (FRET) system by imaging a single cell expressing a standard FRET plasmid with known FRET efficiency (E) and donor-acceptor concentration ratio (RC) (Liu et al., Opt. Express30, 29063 (2022)10.1364/OE.459861). Here we improved the SCC-FRET method (named as Im-SCC-FRET) to simultaneously obtain ß, δ, G, k and the acceptor-to-donor extinction coefficient ratio (ε A ε D), which is a key parameter to calculate the acceptor-centric FRET efficiency (EA), of a FRET system when the range of ß and δ values is set as 0-1. In Im-SCC-FRET, the target function is changed from the sum of absolute values to the sum of squares according to the least squares method, and the initial value of ß and δ estimated by the integral but not the maximum value spectral overlap between fluorophore and filter. Compared with SCC-FRET, the experimental results demonstrate that Im-SCC-FRET can obtain more accurate and stable results for ß, δ, G, and k, and add the ratio ε A ε D, which is necessary for the FRET hybrid assay. Im-SCC-FRET reduces the complexity of experiment preparation and opens up a promising avenue for developing an intelligent FRET correction system.

SCC-FRET: single-cell-based calibration of a FRET system.

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 (ß 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 ß = 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.