Incertitude in the orientation factor, κ2, in Förster-type resonance energy transfer (FRET) has thus far hindered the evaluation of donor-acceptor distances in single-molecule measurements. In part, the confusion arises from directly applying ideas developed for ensemble-averaged experiments. By carefully considering the fundamental difference between single-molecule and bulk measurements, it is shown that, regardless of the excited-state lifetime of the chromophores, the idealized value of 2/3 is a very good approximation (within 10% relative error) for a broad range of parameter space for single-molecule FRET applications. Implications for interpreting and designing single-molecule FRET experiments are also discussed.
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