Notes on Resonance Energy Transfer Dyes

Resonance energy is a process by which a fluorescent molecule (such as phycoerythrin, or PE) can be excited and transfer this energy to a nearby other fluorescent molecule (such as Cy5)--which then emits. This process occurs only when the two dyes are within about 30-50 nm of each other (i.e., covalently linked). As well, the emission spectrum of the donor (in this example, PE) must overlap to some small extent the excitation spectrum of the acceptor (e.g., Cy5).

The resulting "tandem" dye, e.g., Cy5PE can be excited at donor excitation wavelengths but emit at acceptor emission wavelengths. In the case of Cy5PE, this means the molecule can be excited at 488 nm (a wavelength which does not excite Cy5 at all) and emit at 680 nm (at which PE emits extremely little light). In essence, the tandem dyes shift the emission spectrum to lower wavelengths.

Tandem dyes are extremely useful, because they open up entirely new areas of the spectrum. However, they have a downside: every tandem can have slightly different spectral properties, making compensation quite difficult.

In the case of Cy5PE, the degree to which energy is transferred from PE to Cy5 is termed the "efficiency." Ideally, the efficieny would be 1; this never happens. That energy which is not transferred is emitted with a spectrum identical to the PE emission spectrum. Thus, a small part of the emitted light from Cy5PE is directly from PE.

This process can be minimized by maximizing the substitution of the PE molecule with Cy5. The more Cy5 molecules there are, the less PE fluorescence is observed. However, once more than a certain number of Cy5 molecules are on a single PE molecule (about 5), they begin to interact in a way which quenches the resulting Cy5 fluorescence. So, while the PE fluorescence continues to decrease as the substitution increases, so will the Cy5 fluorescence.

With all tandem dyes, a ratio of acceptor to donor must be chosen which suitably quenches the donor fluorescence (i.e., minimize required compensation) but still results in a useful dye (i.e., good acceptor fluorescence). To accomplish this, a titration of donor to acceptor ratios is performed and the resulting conjugates are characterized by FACS (after conjugation to antibodies). Such a titration is critical whenever you embark on synthesis of a tandem for the first time.

Once a good ratio is chosen, then the synthesis is scaled up to reasonably large quantities. This is necessary, because every time a new tandem is made (a new "lot"), the resulting spectral properties will not be identical to the last time. I.e., it is virtually impossible to guarantee the same degree of substitution every time. This in turn means that every lot of a tandem will require slightly or somewhat different compensation when analyzed by FACS.

Examples of the synthesis and characterization of two tandems, Cy7PE and Cy7APC, as well as discussion about optimization of brightness vs. compensation, can be found in:
M Roederer, AB Kantor, DR Parks, and LA Herzenberg: Cy7PE and Cy7APC: Bright new probes for immunofluorescence. Cytometry, 24:191-197 (1996).