Pulse-shaping based two-photon FRET stoichiometry

Daniel C. Flynn, Amar R. Bhagwat, Meredith H. Brenner,  Marcos F. Nunez, Briana E. Mork, Dawen Cai, Joel A. Swanson, and Jennifer P. Ogilvie

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Forster Resonance Energy Transfer (FRET) based measure- ments that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two- photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.