SVC-FRET: Spatial-vector-based calibration on a FRET microscope

Abstract

Accurate quantitative measurement of Förster resonance energy transfer (FRET) in living cells relies critically on precise system calibration parameters. Coullomb et al. introduced QuanTI-FRET, a method for determining system calibration parameters (G and k) through three-dimensional fitting using standard FRET plasmids of known stoichiometry. However, this method requires pre-determined spectral crosstalk coefficients (a and d) from donor-only and acceptor-only samples. To address this limitation, we propose SVC-FRET (spatial-vector-based calibration on a FRET microscope), which utilizes three-channel intensity distributions from three standard FRET plasmids to construct spatial vectors. Through fitting of these vectors under physical constraints, SVC-FRET simultaneously resolves five key parameters—including spectral crosstalk correction parameters ($\beta \approx a$, $\delta \approx d$), system calibration parameters (G, k), and the acceptor-to-donor extinction coefficient ratio (${\epsilon }_A/{\epsilon }_D$). Experimental results demonstrate that SVC-FRET-derived parameters enable reliable quantitative FRET analysis. Compared to QuanTI-FRET, SVC-FRET streamlines experimental workflows by eliminating the need for separate donor/acceptor samples and reduces potential errors from experimental condition inconsistencies, thereby enhancing robustness and measurement reliability.