FRET has become an important tool to investigate the structure and dynamics of proteins and other molecular systems. The recent development of new fluorescent probes and labeling techniques and the advancement in instrumentation has resulted in a rapid proliferation of the technique. FRET is applied to systems of ever increasing size and complexity. This has highlighted some of the difficulties when examining real-life systems. One theoretical challenge involves linking the observed FRET efficiency to the spatial arrangement of the participating fluorophores. Although the relationship between FRET efficiency and fluorophore separation has been well described for a single donor-acceptor pair, a system that contains multiple donors and acceptors cannot be described by this single distance model. Many current and potential applications of FRET involve multiple fluorophores. Our own application of FRET for studying the conformational changes associated with the gating of ion channel proteins has motivated the development of a tool to aid in the analysis and interpretation of FRET in both simple and complex geometries.

ExiFRET is a simple and flexible program that employs a Monte Carlo scheme to calculate FRET between fluorophores distributed in a variety of geometries. The arrangement of fluorophores can range from simple cases such as a random distribution of individual fluorophores to more complex cases of linked donor-acceptor pairs or fluorophores arranged in regular geometries (eg. pentamers), both in 2 and 3 dimensions. ExiFRET is highly flexible and allows the user to control the geometry of the fluorophores through a series of parameters in the input files (see User guide). The program also enables the user to define their own geometries characteristic to the system under instigation to ensure the simulation emulates the experimental conditions.
Once the fluorophore geometries are assigned, the response of the system to incident photons is determined from the probability of each donor fluorescing or transferring energy to an acceptor. A large number of geometries and photons should be used to gain statistically reliable results. The output produced by the program can then be used to generate plots that help in the analysis and interpretation of FRET experiments (see examples). Example input files for the Figures found in the newest ExiFRET publication can be found in examples 2 .

The following is a short overview of the steps involved in the Monte Carlo calculation scheme

ExiFRET was developed in the Corry Research Group at the University of Western Australia. We are now located at the Australian National University.