Multiplexed FRET to image multiple signaling events in live cells

Abstract

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

FIGURE 1

Multiplexed FRET microscope. Spectral ratiometric images of Cameleon were acquired by exciting ECFP with a continuous wave blue laser and resolving the fluorescence into two spectral channels. For FLIM measurements an ultrafast supercontinuum excitation source (Fianium, UK) was spectrally filtered for TagRFP excitation. TagRFP fluorescence was imaged onto a GOI (gated optical intensifier (Kentech Instruments, model HRI) and time-gated fluorescence images were recorded on a Hamamatsu ORCA-ER charge coupled device camera.

FIGURE 2

FRET constructs and spectral channels used. (A) Binding of calcium to calmodulin domain of the YCAM 3.6 Cameleon results in a conformational change and FRET from ECFP to Venus. Activation of mPlum labeled H-Ras (exchange of GDP for GTP catalyzed by guanonucleotide exchange factor (GEF)) results in recruitment of Tag-RFP labeled Raf-Ras Binding Domain to the membrane and FRET between TagRFP/mPlum. (B) Absorption and emission spectra of the 4 fluorophores. Shaded areas indicate excitation and emission bands used for imaging multiplexed FRET. Filters are given in the Supplementary Material, Data S1.

FIGURE 3

Multiplexed FRET imaging of calcium flux and Ras activation. (A) Spectral ratiometric images of Cameleon at time points shown (times in seconds). Epidermal growth factor was added 10s from the start. (B) Lifetime maps of TagRFP at time points shown (top row) and lifetime merged with intensity (bottom row). (C) Intensity traces in the Venus and ECFP spectral channels from a region of interest in the image and mean lifetime of TagRFP from a region in the membrane. Details of cell culture and plasmid construction are given in the Supplementary Material, Data S1.