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 u sing sensitized acceptor decay measurements. In these measurements a long m icrosecond lifetime of the lanthanide donor and a short nanosecond Lifetime of the acceptor allow elimination of a signal from the unquenched donor. T herefore, the decay of sensitized acceptor emission reflects decay properti es 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 a cceptor signal is dependent on the resonance energy transfer rate constant. Thus, in the case where there are two or more populations of donors with d ifferent energy transfer rate constants, the relative amplitudes of corresp onding decay components observed in sensitized acceptor emission do not rep resent the relative populations of the donors. We use simulations to show t hat these effects can be very significant. A minor population of donors wit h a high rate of energy transfer can produce sensitized acceptor decay whic h is dominated by a decay component corresponding to this minor donor popul ation. Using a simple experimental system of rapid diffusion Limit energy t ransfer between a europium chelate and Cy5 acceptor we show that the predic ted dependency of sensitized acceptor decay amplitude on the energy transfe r rate is indeed observed. We suggest that the relative importance of decay components observed in sensitized acceptor emission should be evaluated af ter an appropriate correction of their values such that they properly refle ct possible different populations of donors. We describe a method to perfor m such correction. (C) 2001 Academic Press.