The inner workings of living cells are made up of networks of proteins binding and releasing each other. A powerful approach to capture the orchestration of these events is imaging of Förster Resonance Energy Transfer (FRET) between two proteins tagged with donor and acceptor fluorescent proteins. Since FRET occurs over a length scale similar to the size of most proteins (~ 5 nanometers), FRET signals can be used to image protein interactions in living cells. However, obtaining this rich information from ‘intermolecular FRET’ microscopy requires careful microscope calibration and optics to separate overlapping fluorescence signals between the donor and acceptor’s spectral signatures in order to quantify how many molecules are engaged in FRET. A frontier for FRET microscopy is to study protein interactions in tissues by two-photon microscopy, but a challenge for this approach is to sequentially and selectively excite the donor and acceptor fluorophores.

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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