Accurate Use of Single Molecule Fluorescence Correlation Spectroscopy to Determine Molecular Diffusion Times

Abstract

We report on several aspects of experimental method and autocorrelation analysis of fluorescence bursts from single molecules freely diffusing in solution through a focused light beam. We show that the commonly occurring inclusion of zero lag times and use of long sampling times when performing software autocorrelation leads to inaccurate values for molecular diffusion times and we compare these data with those obtained with hardware autocorrelation. We also report values for the diffusion coefficient of two dye molecules measured independently by NMR which can be used as reference values for single molecule fluorescence correlation spectroscopy (Rhodamine 6G 2.9 ± 0.7 × 10-10m2s-1 and BODIPY FL CASE 3.8 ± 1.0 × 10-10m2s-1).