Using the apparent fluorescence stoichiometry ratio, , it is possible to identify bursts from molecules with either only active donor (i.e., ≈ 1) or only active acceptor (i.e., ≈ 0) fluorophores. The apparent mean transfer efficiency, , of the donor-only subpopulation is used to determine the correction factor for cross-talk, . The apparent mean fluorescence stoichiometry ratio, , associated with the acceptor-only subpopulation is used to determine the correction factor for direct excitation, (due to background correction, Ê values for the acceptor-only bursts extend beyond the range shown). With pulsed interleaved excitation (PIE), time-correlated single-photon counting, and four-channel detection, it is also possible to obtain time-resolved fluorescence intensity and anisotropy decay plots from the parallel and perpendicular emission of the donor-only subpopulation after donor excitation (i.e., ∥ and ⊥ ) and the parallel and perpendicular emission of the acceptor-only subpopulations after acceptor excitation (i.e, ∥ and ⊥ ). This information is then used to determine the fluorescence lifetimes of donor and acceptor fluorophores and the time-resolved and the steady-state anisotropies (rss) of the fluorophores.