Note that the optimal set of filters for FRET need not necessarily use the same exciter and/or emitter filters optimized for measurements of the individual donor and acceptor fluorophores. For example, Figure 4 shows the same set from Figure 3 for CFP-YFP FRET, except here the YFP emitter has been replaced by an emitter optimized for FITC fluorescence (BrightLine FF506-Em02). Because one need not worry about placing an exciter filter for the acceptor very near the peak of the acceptor absorption, it is possible to use an emitter that fully spans the acceptor emission peak, thus providing a substantially higher FRET signal. However, a wider-band emitter generally also allows more background noise.
Figure 4: Replacement of the YFP emitter filter - optimized for high signal-to-noise analysis of YFP alone - with a FITC emitter filter (BrightLine FF506-Em02) leads to more efficient (but also somewhat higher-noise) measurement of acceptor emission.
The imaging performance of optical filters used for FRET is also critical, since for accurate measurements a ratio of the intensities measured on two separate images must be computed. If there is any relative movement of the images (even for a perfectly stationary sample), the ratio calculation will be distorted. The simplest and most accurate way to avoid this problem is to use filter sets which cause no "pixel shift," or relative variation of the beam deviation caused by non-zero wedge angles of the dichroic and emission filters. Semrock's BrightLine ZERO™ filter sets offer certified zero-pixel-shift performance to eliminate the pixel-shift problem. This unique performance is made possible by the patented BrightLine filter technology (U.S. Patent No. 6,809,859 and pending) that is based on a single glass substrate coated by durable, hard coatings.
Selected references for further reading on FRET:
- J. R. Lakowicz, "Principles of Fluorescence Spectroscopy" (Kluwer Academic / Plenum Publishers, New York, 1999).
- Paul R. Selvin, "The Renaissance of Fluorescence Resonance Energy Ttransfer," Nature Structural Biology, Vol. 7, No. 9, pp. 730-734 (2000).
- Handbook of Fluorescent Probes and Research Products, Molecular Probes, www.probes.com/handbook.
