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 (β 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 (R_C) (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 β, 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 (E_A), of a FRET system when the range of β 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 β 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 β, 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.