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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.
Highlights
IntroductionForster (or equivalently “fluorescence”) Resonance Energy Transfer (FRET) based microscopy uses nonradiative energy transfer from one molecule (donor) to another (acceptor) to provide information about molecular interactions within biological systems [1,2]
Forster Resonance Energy Transfer (FRET) based microscopy uses nonradiative energy transfer from one molecule to another to provide information about molecular interactions within biological systems [1,2]
We demonstrate that intensity-based one-photon FRET stoichiometry approaches can be adapted to two-photon FRET applications using pulse-shaping of a broadband titanium:sapphire oscillator
Summary
Forster (or equivalently “fluorescence”) Resonance Energy Transfer (FRET) based microscopy uses nonradiative energy transfer from one molecule (donor) to another (acceptor) to provide information about molecular interactions within biological systems [1,2]. Often referred to as a “spectroscopic ruler”, the FRET efficiency is proportional to 1/r6, where r is the intermolecular distance. This results in energy transfer only when the donor and acceptor molecules are within close proximity (1–10 nm) and can provide information on a wide range of molecular interactions [2,3,4,5,6]. Several approaches have been taken to enable quantitative FRET measurements. These include methods that employ various combinations of multiple excitation wavelengths and detection channels, as well as spectrally-resolved and fluorescence lifetime imaging (FLIM) approaches
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