Stern-Volmer modeling of steady-state Forster energy transfer between dilute, freely diffusing membrane-bound fluorophores.

Two different metrics are used to assess Forster resonance energy transfer (FRET) between fluorophores in the steady state: (i) acceptor-quenching of donor fluorescence E (also known as transfer efficiency) and (ii) donor-excited acceptor fluorescence F(A) (Dex). While E is still more widely used, F(A) (Dex) has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under dilute-probe conditions. We have also discovered a dilute-regime correspondence between our Stern-Volmer expression for E and Wolber and Hudson's series approximation for steady-state Forster quenching in two dimensions (2D). This novel correspondence allows us to interpret each of our 2D quenching constants in terms of both (i) an effective Forster distance and (ii) two maximum acceptor-concentration limits, each of which defines its own useful experimental regime. Taken together, our results suggest a three-step strategy toward designing more effective steady-state FRET experiments for the study of biomembranes.