After energy transfer occurs from donor to acceptor, the acceptor fluorophore is excited to its fluorescence emission state. Because the observed rate of fluorescence emission from the acceptor is rate-limited by energy transfer from donor to acceptor, the quantitative measurement of FRET emission can therefore provide an inferred measurement of distance using the equation above. Accurate FRET determination generally involves comparison of the donor and donor-acceptor fluorescence emission intensities in samples with and without the acceptor present. A ratio measurement is necessary because, as Figure 1 demonstrates, there is typically overlap between the donor and acceptor emission spectra, thus making it difficult to determine with a single measurement exactly what fraction of the fluorescence measured with an acceptor emission filter derives from only the acceptor. Fluorescence lifetime measurements provide more direct results for the energy transfer rate, are not susceptible to concentration variations, and can be made using time domain or phase modulation lifetime measurement techniques. These types of measurement can also provide information regarding conformational changes due to molecular interactions based on further detailed knowledge of the bound and/or conjugated FRET fluorophore pair systems.
Application conditions for observing FRET are often characterized by extremely low molecular fluorophore concentrations thus requiring detection of very faint fluorescence emission levels. Optical fluorescence filters are a critical part of an optical system for FRET detection. Semrock BrightLine fluorescence filters offer the highest possible transmission for maximizing the FRET emission signal, as well as carefully optimized deep blocking out of the transmission passbands, for maximum possible signal-to-background ratios (highest contrast).
Figure 2 shows the transmission spectra for a filter set optimized for measuring CFP (BrightLine CFP-2432A set), and Figure 3 shows the same exciter and dichroic filter spectra with the spectrum of a YFP emitter filter (BrightLine FF520-Em01). The set in Figure 2 is used to quantify the fluorescence from the donor alone, while the set in Figure 3 is used to quantify the FRET fluorescence from the acceptor. These two measurements may
be made sequentially with two different filter cubes mounted in a fluorescence microscope turret, each of which has identical exciter and dichroic filters, but different emitter filters. Alternatively, because samples in live-cell imaging applications are often in motion, more rapid filter changes can be achieved with an emitter filter wheel.
Figure 2: CFP exciter, dichroic, and emitter filters (BrightLine FF458 set) for quantitative measurement of donor emission.
Figure 3: CFP exciter and dichroic filters with YFP emitter filter (BrightLine FF520-Em01) for quantitative measurement of acceptor emission.
