Figure 1.

Molecule with physical states σ₁ and σ₂ labeled with a donor/acceptor pair. In the absence of blinking, σ₁ is associated with a brighter donor and low FRET efficiency, while σ₂ is associated with a brighter acceptor and high FRET efficiency. For well separated distributions, apparent efficiency suffices to distinguish between σ₁ and σ₂. However, in the presence of blinking, apparent distributions alone may lead to misinterpretation of the molecule’s state since low efficiencies may be observed while the molecule is at σ₂ and, vice versa, high efficiencies may be observed while the molecule is at σ₁.