Abstract
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 f1 and f2 by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at f1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f1 but also at the beat frequency Δf: = |f1 − f2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.
Highlights
Since the ensemble contains donor acceptor pairs engaged in Förster Resonance Energy Transfer” (FRET), the released donor fluorescence is modulated at f1 and at the beat frequency Δf: = f1 − f2
They concluded from their experiments that the transfer of kinetic energy during the atomic collisions of the second kind, which at the same time resulted in the transfer of quantum energy, can—if required—cover missing quantum energy for the previously-described energy transfer from gas atoms of one type to the gas atoms of a second type
It is today commonly referred to as the “Förster Resonance Energy Transfer”, abbreviated “FRET”. He concluded in mathematical terms that, other than during the energy transfer via a mediating luminescent photon, or radiative energy transfer, FRET only can occur if the two chromophores are quasi-co-localized and he derived that the rate of the energy transfer by means of FRET is dependent on the sixth power of the distance between donor and acceptor [6,7]
Summary
In 1923, based on theoretical considerations by Klein and Rosseland [1], Franck and Cario described their observation that energy stored in the electronic configuration of excited gas atoms of one type can be transferred to gas atoms of another type, given that the amount of energy stored in the excited gas atoms of the first type is suitable for the excitation of the gas atoms of the second type [2]. It is today commonly referred to as the “Förster Resonance Energy Transfer”, abbreviated “FRET” He concluded in mathematical terms that, other than during the energy transfer via a mediating luminescent photon, or radiative energy transfer, FRET only can occur if the two chromophores are quasi-co-localized and he derived that the rate of the energy transfer by means of FRET is dependent on the sixth power of the distance between donor and acceptor [6,7]. Quantitative interpretation of FRET efficiencies in suitable regions of interest (ROI) in digitized microscopic images is equivalent to measuring relative or, given a sufficient calibration, even absolute distances in domains surpassing the resolution limit of conventional or even confocal light microscopes by up to two orders of magnitude. The aim of this proposal is to present a new approach for fully quantitative, rapid, and non-destructive FRET measurements
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