Proposal of a New Method for Measuring Förster Resonance Energy Transfer (FRET) Rapidly, Quantitatively and Non-Destructively

The process of radiationless energy transfer from a chromophore in an excited electronic state (the "donor") to another chromophore (an "acceptor"), in which the energy released by the donor effects an electronic transition, is known as "Förster Resonance Energy Transfer" (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies ƒ(1) and ƒ(2) by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at ƒ(1), and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at ƒ(2). Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at ƒ(1) but also at the beat frequency Δƒ: = |ƒ(1) - ƒ(2)|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.