Pulse-shaping multiphoton FRET microscopy.

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them. Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of two-photon FRET. This work paves the way for two-photon FRET stoichiometry.