Abstract
A concentration as low as 0.1 molecule per observation volume may not be small enough for single-molecule Förster resonance energy transfer (FRET) efficiency measurements of molecules diffusing through a laser spot. This result follows from a rigorous theory that takes many molecules into account. We consider the distributions of the number of photons (photon counting histograms) and show that multiple-molecule effects are pronounced at large photon counts even at low concentrations. FRET efficiency distributions reveal multiple-molecule effects at large threshold values. This might be misinterpreted as multiple conformational states. Multiple-molecule effects strongly depend on the brightness of fluorophores. A simple test is suggested to determine parameters for which the single-molecule description is applicable.
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