title: Concentration effects in "single-molecule" spectroscopy.
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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. irinag@niddk.nih.gov
authors.forename: Irina V
authors.initials: IV
authors.lastname: Gopich
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017785:Photons