Proposal of a new method for measuring förster resonance energy transfer (FRET) rapidly, quantitatively and non-destructively

Abstract

The process of radiationless energy transfer from a chromophore in an excitedelectronic state (the "donor") to another chromophore (an "acceptor"), in which the energyreleased by the donor effects an electronic transition, is known as "Förster ResonanceEnergy Transfer" (FRET). The rate of energy transfer is dependent on the sixth power ofthe distance between donor and acceptor. Determining FRET efficiencies is tantamount tomeasuring distances between molecules. A new method is proposed for determining FRETefficiencies rapidly, quantitatively, and non-destructively on ensembles containing donoracceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors andacceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle 1/2and frequencies f 1 and f 2 by electro-optic modulators. In an ensemble exposed to these laserbeams, the donor excitation is modulated at f 1, and the acceptor excitation, and thereforethe degree of saturation of the excited electronic state of the acceptors, is modulated at f 2.Since the ensemble contains donor acceptor pairs engaged in FRET, the released donorfluorescence is modulated not only at f1 but also at the beat frequency Δf:=|f 1 - f 2 |. Thedepth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicatesthe FRET efficiency. © 2012 by the authors; licensee MDPI, Basel, Switzerland.