Förster resonant energy transfer (FRET)
FRET is a well-established fluorescence-based technique to detect and quantify biomolecular interactions, including readouts of genetically expressed FRET biosensors. FRET measurements require the biomolecules of interest to be labelled with complementary fluorophores, such that the emission spectrum of one fluorophore (called the “donor”) overlaps with the excitation spectra of the second fluorophore (the “acceptor”). When these complementary fluorophores are in close proximity (<~10 nm) there can be an efficient energy transfer process resulting in a reduction in emission from the donor fluorophore and fluorescence emission of the acceptor that is only excited via the energy transfer. The efficiency of this energy transfer scales inversely with the 6th power of the fluorophore separation and is effectively negligible for donor-acceptor distances >~10 nm. Thus detection of FRET is a strong indication of interaction of the labelled biomolecules. Using genetically expressed fluorophores (such as CFP with YFP or GFP with mCherryFP) to label specific proteins, FRET is widely used to map protein-protein interactions in live cells and to readout FRET biosensors, which are molecular constructs that are labelled with both donor and acceptor fluorophores.
FRET can be detected through a wide range of changes in fluorescence parameters of donor of acceptor fluorophores but the two most common FRET readouts are spectral ratiometric FRET and fluorescence lifetime imaging (FLIM). Spectral ratiometric FRET measures the fluorescence intensity of the donor and acceptor emission. The resulting FRET ratio generally provides a qualitative readout of FRET and requires careful calibration to account for spectral cross-talk and other potential artefacts, particularly when the relative donor and acceptor fluorophore concentrations are unknown. Spectral ratiometric FRET is most robust when applied to single molecule FRET biosensors. With FLIM, only the donor emission is measured and FRET is indicated by a decrease in the donor fluorescence lifetime since the energy transfer provides an additional relaxation pathway for the excited donor fluorophores. Fluorescence lifetime-based measurements are therefore independent of donor/acceptor stoichiometry and also of spectral properties of the sample or instrumentation. Furthermore, by fitting the donor fluorescence decay profiles to a double exponential decay model, it is possible to determine the fraction of the donor fluorophore population that is undergoing FRET (and therefore the fraction of labelled biomolecules that are interacting).
The Photonics Group at Imperial College London, we have developed a range of instruments for FLIM, including microscopes, endoscopes and optical tomography systems, and we have developed open source software tools for FLIM data acquisition and analysis. For practical FLIM with rapid image acquisition and low phototoxicity that is compatible with live cell imaging, we have focussed on wide-field time gated FLIM using a gated optical intensifier (GOI). This approach provides robust FLIM data acquisition that is sufficiently rapid to enable automated multiwell plate FLIM for high content analysis, including FLIM/FRET assays and label-free readouts of cellular autofluorescence to report changes in cell metabolism. We have also implemented FLIM in laser scanning microscopes utilising time-correlated single photon counting (TCSPC), including microscopes combining TCSPC with polarisation-resolved detection for studies complementing FLIM/FRET data with time-resolved fluorescence anisotropy measurements and laser scanning confocal FLIM endomicroscopy for in vivo FLIM/FRET studies to quantify intracellular chemotherapeutic drug-target binding.
Our open source FLIM data analysis software, FLIMfit, is applicable to both wide-field time-gated FLIM and TCSPC data and provides global analysis capabilities.
For FLIM data acquisition, TCSPC FLIM is available with a range of commercial microscopes or can be implemented as an commercially available upgrade with proprietary software. Wide-field time-gated FLIM can be implemented using our open source FLIM software to control the instrument and the data acquisition. This is available as a MicroManager plug-in for manual FLIM microscopy, as a module for our new MicroManager-based HCA platform or FLIM HCA can be implemented using our standalone FLIM HCA MicroManager plug-in that is supported by detailed instructions and hardware component lists in this JoVE paper.