How do I optimize FRET?

To maximize FRET efficiency, it's essential to choose a FRET pair with red-shifted emission spectra and enhanced properties such as quantum yield (QY), extinction coefficient (EC), and spectral overlap. FRET efficiency (E) is inversely proportional to the sixth power of the distance between the two fluorophores and is effective over distances shorter than 10 nm. The Forster distance should align with the experimental goals and the mechanism of donor-acceptor interaction. Linking the donor and acceptor to the same large molecule structure or separately labeling molecules with donor and acceptor are typical methods. These then interact through specific binding on a shared scaffold or surface. Control experiments are crucial to ensure observed changes in donor and acceptor emission result from FRET rather than other factors. A basic control is a no-FRET reference state with only the donor, but additional controls can be beneficial. One should also design the experiment based on if it relies on fluorescence intensity or lifetime and determine whether this assessment applies to the donor, the acceptor, or both.