Single distance between donor and acceptor

The Forster resonance energy transfer can be used as a spectroscopic ruler in the range of 10-100 A. The distance between the donor and acceptor molecules should be constant during the donor lifetime, and greater than about 10 A in order to avoid the effect of short-range interactions. The validity of such a spectroscopic ruler has been confirmed by studies on model systems in which the donor and acceptor are separated by well-defined rigid spacers. Several precautions must be taken to ensure correct use of the spectroscopic ruler, which is based on the use of Eqs (9.1) to (9.3)  

This section deals with a single donor-acceptor distance. Let us consider first the case where the donor and acceptor can freely rotate at a rate higher than the energy transfer rate, so that the orientation factor k2 can be taken as 2/3 (isotropic dynamic average). The donor-acceptor distance can then be determined by steady-state measurements via the value of the transfer efficiency (Eq. 9.3)  

Three steady-state methods can be used to determine the energy transfer efficiency. In the following description of these methods, the fluorescence intensity is indicated with two wavelengths in parentheses the first one is the excitation wavelength, and the second is the observation wavelength. Because the characteristics of the donor and/or acceptor are measured in the presence and in the absence of transfer, the concentrations of donor and acceptor and their microenvironments must be the same under both these conditions. 

Steady-state method 1 decrease in donor fluorescence Transfer from donor to acceptor causes the quantum yield of the donor to decrease. The transfer efficiency is given by 

Because only the relative quantum yields are to be determined, a single observation wavelength is sufficient and the latter is selected so that there is no emission from the acceptor1 . Then, Eq. (9.7) can be rewritten in terms of absorbances at the excitation wavelength 7d and fluorescence intensities of the donor in the absence and presence of acceptor  

As regards the orientation factor k, it is usually taken as 2/3, which is the isotropic dynamic average, i.e. under the assumptions that both donor and acceptor transient moments randomize rapidly during the donor lifetime and sample all orientations. However, these conditions may not be met owing to the constraints of the microenvironment of the donor and acceptor. A variation of from 2/3 to 4 results in only a 35% error in r because of the sixth root of 

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