title: Pulse-shaping based two-photon FRET stoichiometry.
abstract: Förster Resonance Energy Transfer (FRET) based measurements 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.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Flynn
authors.forename: Amar R
authors.initials: AR
authors.lastname: Bhagwat
authors.forename: Meredith H
authors.initials: MH
authors.lastname: Brenner
authors.forename: Marcos F
authors.initials: MF
authors.lastname: Núñez
authors.forename: Briana E
authors.initials: BE
authors.lastname: Mork
authors.forename: Dawen
authors.initials: D
authors.lastname: Cai
authors.forename: Joel A
authors.initials: JA
authors.lastname: Swanson
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: Ogilvie
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D036641:Microscopy, Fluorescence, Multiphoton; D008962:Models, Theoretical; D055620:Optical Phenomena; D017785:Photons; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins
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