A number of fluorescent proteins have been described in recent years. For example, green fluorescent protein (GFP) is a native fluorescent molecule found in the jellyfish Aequorea victoria. Genetic modification of green fluorescent protein has led to variants, such as enhanced green fluorescent protein (EGFP), that exhibit modified fluorescence as compared to native green fluorescent protein. The variants are sometimes referred to as mutants of native green fluorescent protein.
In some cases, these variants exhibit fluorescence excitation efficiencies that are greater than the native green fluorescent molecule. Alternatively, or in combination, the variants may exhibit fluorescence at wavelengths greater or less than the native green fluorescent protein. For example, some variants include mutations within the coding sequence of GFP and these mutations lead to wavelength shifts of the GFP fluorescence into, e.g., the yellow and blue regions of the visible spectrum. Various pairs of these variants, e.g., enhanced yellow fluorescent protein (YFP) and enhanced cyan fluorescent protein (CFP), exhibit peaks for fluorescence excitation and fluorescence emission that are generally distinct from one another. Generally, however, some degree of spectral overlap remains.
Fluorescence resonance energy transfer (FRET) is a useful biophysical technique that requires some overlap between the fluorescence emission spectrum of a first molecule and the fluorescence excitation spectrum of another molecule. In FRET, a sample including a first fluorescent molecule (a FRET donor) and a second fluorescent molecule (a FRET acceptor) is irradiated with light. The donor and acceptor may interact with the light in several ways. First, the FRET donor may absorb light and emit fluorescence (referred to as donor fluorescence). Second, the FRET acceptor may absorb light and emit fluorescence (referred to as acceptor fluorescence). Additionally, the FRET donor can absorb light and transfer the energy gained to the FRET acceptor. The FRET acceptor generally emits light (FRET emission) at wavelengths characteristic of the acceptor fluorescence.
FRET requires that the FRET donor and FRET acceptor molecule be within close proximity, e.g., less than about 10 nanometers of one another. Thus, FRET can be used to probe for molecular interactions of two or more molecules within living cells. For example, interactions between molecules, e.g., proteins, receptors, and substrates, respectively labeled with a FRET-donor and FRET-acceptor can be probed (see, e.g., Clegg, “Fluorescence Resonance Energy Transfer,” In: Fluorescence Imaging Spectroscopy and Microscopy, Wang and Herman, Eds., Wiley, New York, 1996, vol. 137, pp 179-251).
The fluorescence emission spectrum of CFP, for example, overlaps with the fluorescence excitation spectrum of YFP. Thus, CFP (the FRET donor) and YFP (the FRET acceptor) may exhibit FRET if positioned closely enough and upon the absorption of light by CFP.
To determine whether FRET has occurred, however, the FRET emission must be discriminated from the donor fluorescence and acceptor fluorescence. Several approaches are possible. One approach takes advantage of the fact that the wavelengths of the fluorescence spectra of the FRET donor and FRET acceptor are significantly different from one another. Additionally, the fluorescence excitation spectra of the FRET donor and FRET acceptor should also be significantly different. FRET emission is determined by detecting emission with various combinations of irradiating wavelengths and detection wavelengths. However, because some overlap exists between the excitation and emission spectra, calibration is required to determine the amount of FRET. In general, calibration includes measuring emission from a calibration sample having known regions of donor fluorescence, known regions of acceptor fluorescence, and known regions of FRET emission. The emission from the three regions can be used to calibrate an instrument for determining the amount of FRET observed from a sample, e.g., a cell.
In another approach, FRET emission is distinguished from donor fluorescence and acceptor fluorescence by measuring the lifetime of the detected emission. Typically, the transfer of energy from the FRET donor to the FRET acceptor shortens the lifetime of the donor fluorescence. However, because the donor fluorescence lifetime is also sensitive to environmental factors, calibration is generally required to determine whether FRET has occurred. Calibration generally involves determining the lifetime of donor fluorescence both in the presence and absence of a FRET acceptor. The lifetimes can be used to calibrate an instrument for determining the amount of FRET observed from a sample.