Concentration effects in "single-molecule" spectroscopy.
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.
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017785:Photons