Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes

Lanthanide chelates used as donors offer several advantages over classical fluorescence probes in resonance energy transfer distance measurements. One of these advantages is that energy transfer can be conveniently measured using sensitized acceptor decay measurements. In these measurements a long microsecond lifetime of the lanthanide donor and a short nanosecond lifetime of the acceptor allow elimination of a signal from the unquenched donor. Therefore, the decay of sensitized acceptor emission reflects decay properties of the donor engaged in energy transfer. The purpose of this work is to point out the importance of the fact that the amplitude of the sensitized acceptor signal is dependent on the resonance energy transfer rate constant. Thus, in the case where there are two or more populations of donors with different energy transfer rate constants, the relative amplitudes of corresponding decay components observed in sensitized acceptor emission do not represent the relative populations of the donors. We use simulations to show that these effects can be very significant. A minor population of donors with a high rate of energy transfer can produce sensitized acceptor decay which is dominated by a decay component corresponding to this minor donor population. Using a simple experimental system of rapid diffusion limit energy transfer between a europium chelate and Cy5 acceptor we show that the predicted dependency of sensitized acceptor decay amplitude on the energy transfer rate is indeed observed. We suggest that the relative importance of decay components observed in sensitized acceptor emission should be evaluated after an appropriate correction of their values such that they properly reflect possible different populations of donors. We describe a method to perform such correction.