Figure 2.

Three-color FRET using alternating excitation. (a) Photon trajectories of three fluorophores detected after alternating laser excitation of the donor (D) (485 nm, blue dashed line) and acceptor 1 (A1) (595 nm, yellow dashed line) (40 MHz repetition rate). For each photon, the absolute arrival time (t) and the relative delay time (δt) between the laser pulse and the photon arrival is recorded. Photons detected after D excitation (index i) and A1 excitation (index j) can be separated (second and third rows) using ~ 25 ns difference in their delay times. (b) Definition of the FRET efficiencies between dye pairs. E₁ is the energy transfer efficiency from D to A1 in the absence of acceptor 2 (A2), E₂ is the transfer efficiency from D to A2 in the absence of A1, and E₁₂ is the transfer efficiency from A1 to A2. θ is the angle between two distance vectors r₁ and r₁₂. r₂ = r₁ + r₁₂. (c) Excited state dynamics in three-color FRET. After donor excitation (DA₁A₂ → D*A₁A₂), the donor decays to the ground state radiatively (green rippled arrow) or non-radiatively (dashed arrow), or the energy is transferred to either A1 (DA₁*A₂) or A2 (DA₁A₂*), with the energy transfer rates of k_ET1 and k_ET2. A1 excited state decays to the ground state or the energy is further transferred to A2 with the rate of k_ET12. After A1 excitation, A1 decays to the ground state or the energy is transferred to A2. k_D, k_A1, and k_A2 are the sums of the radiative and non-radiative relaxation rates of the excited state of D, A1 and A2, respectively, which are equal to the inverse of the excited state lifetimes in the absence of the energy transfer, ${\tau }_{\text{D}}^0$, ${\tau }_{\text{A}1}^0$, and ${\tau }_{\text{A}2}^0$, respectively.