Dynamic Monte Carlo Simulations to Model FRET and Photobleaching in Systems with Multiple Donor−Acceptor Interactions

Monte Carlo simulations were employed to model FRET in systems that included interactions between multiple donors and acceptors and photobleaching. Simulations revealed that pixel-to-pixel variations in microscopy experiments can be due both to variations in the probe distribution and to photon noise, depending on the intensity and probes per detection pixel. The Monte Carlo simulations were used to describe fluorescence experiments on (single) actin filaments using a conventional fluorescence microscope. The filaments were labeled with TRITC at phalloidin and Cy5-maleimide at cys374 and the donor−acceptor distances were taken from literature. The images show a large pixel-to-pixel variation in the fluorescence intensity and suffer from photobleaching. In experiments, the main source of noise was the probe distribution. The acceptor suffered from rapid photobleaching, which resulted in an increase of the donor intensity in time and hampers the straightforward determination of the energy transfer efficiency. The photobleaching of both the donor and the acceptor have been included in the simulations, enabling accurate modeling of the time course of the fluorescence intensities of both donor and acceptor and the energy transfer efficiency.