Simulation Of The Measurement Error Of Independent Emission-Spectral Unmixing Fret

Independent emission- spectral unmixing fluorescence resonance energy transfer, Iem-spFRET, is a novel and powerful tool for measuring FRET efficiency in real time. In this paper, we simulate the measurement error of the Iem-spFRET by introducing random noise in sample data, donor fingerprint, and acceptor fingerprint. The random noise intensity is set from 0.0005 to 0.0025, corresponding to 5% - 25% of the maximum donor fingerprint intensity. The simulated results show the effect of random noise on apparent FRET efficiency (E-fD) is less than on receptor-to-donor concentration ratio (R-c). Random noise with 10% maximum donor fingerprint intensity only leads to 0.33% variation of when the noise is added to both sample and fingerprints. These results indicate that Iem-spFRET is a robust method and could be applied on cases with weak FRET signal.