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.

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

“…By using CFP and YFP as donor and the same red acceptor (tHcRed), FLIM of CFP and YFP donors allow the two different FRET signals to be distinguished [51]. Combination of FLIM-FRET of a red-shifted TagRFP/mPlum pair with ratio imaging of a CFP/Venus pair allows maximal the spectral separation while, at the same time, overcoming the low quantum yield of the far-red acceptor mPlum [52]. The two last examples alleviated the spectral bleedthrough but not the limitation associated with multiple excitations.…”

Single Molecule Tools Elucidate H2A.Z Nucleosome Composition

“…FLIM can be used to study FRET interactions because energy transfer from the donor to acceptor (targeting variants and modifications) will result in a decrease in the donor's fluorescence lifetime. FLIM-FRET is an excellent technique to monitor co-presence distances (1-10 nm) between fluorophore pairs because the change in lifetime of the donor can be analyzed independently of acceptor emission (Bastiaens and Squire, 1999;Peter et al, 2005;Suhling et al, 2005;Grant et al, 2008;Vidi et al, 2008). In FLIM-FRET measurements, fluorescence lifetimes were obtained from TCSPC decay curves fitted by an exponential equation using the SymphoTime software (PicoQuant).…”

High-speed multifocal array scanning using refractive window tilting

“…It has found many applications in life sciences research [10] and drug discovery [11]. One of the major applications is to detect protein-protein interactions by using FLIM to measure Förster resonance energy transfer (FRET).…”

“…One of the major applications is to detect protein-protein interactions by using FLIM to measure Förster resonance energy transfer (FRET). In FLIM-FRET, the fluorescence lifetime is decreased when the excitation energy of a "donor" fluorophore is non-radiatively transferred to a nearby "acceptor" fluorophore [10][11][12]. Energy transfer is only efficient at very short distances, typically less than 10 nm.…”