In this work, Flynn and co-workers, develop a new approach to enable two-photon imaging of FRET with a single broadband laser. Their approach takes advantage of the large spectral bandwidth (~300nm) that is available in an ultrafast laser with femtosecond pulses. By using pulse-shaping, Flynn et al. tailor laser pulses to preferentially excite the donor or acceptor fluorophore – thereby making a single, expensive laser, do the work of two lasers tuned to excite donor or acceptor fluorophores. Unlike one photon microscopy, the spectral bandwidths of two-photon pulses are much broader, leading to undesirable partial excitation of the donor or acceptor. Flynn et al. overcome this limitation by revising the mathematics of the FRET Stoichiometry approach to account for partial excitation of donor or acceptor during their sequential excitations. This modification enabled Flynn and colleagues to successfully image FRET between fluorescent proteins using pulse-shaping two-photon excitation from a single laser. This work advances FRET microscopy by allowing preferential donor and acceptor excitation from a single two-photon laser, and opens up new possibilities for probing protein networks within thicker specimens such as large cells and tissues. Furthermore, pulse-shaping increases the number of ways to control the excitation spectrum of ultra-fast lasers and would potentially allow preferential excitation for multifluorophore FRET experiments.
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