SCC-FRET: single-cell-based calibration of a FRET system.
Opt Express
; 30(16): 29063-29073, 2022 Aug 01.
Article
en En
| MEDLINE
| ID: mdl-36299090
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 (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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Transferencia Resonante de Energía de Fluorescencia
/
Microscopía
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Opt Express
Asunto de la revista:
OFTALMOLOGIA
Año:
2022
Tipo del documento:
Article