Threshold-free evaluation of near-surface diffusion and adsorption-dominated motion from single-molecule tracking data of single-stranded DNA through total internal reflection fluorescence microscopy

Total internal reflection fluorescence (TIRF) microscopy enables the single-molecule observation in liquid near substrate surface. However, the evaluation of the diffusion from their individually-tracked positions entails the difficulty in the treatment of molecular adsorption on the substrate. We propose a novel technique to evaluate them, and two types of near-surface Brownian motion were determined for DNA. One is the diffusion near glass surface, and the other is the adsorption-dominated motion, which is also found to be diffusive rather than anchored to the substrate. Our technique does not require the threshold values for the distinction, and even the transition between them can be captured. Objective distinction of Brownian motion with and without adsorption does not require the adsorption-free sample preparation. It is also useful for the characterization of adsorption/desorption kinetics. Our method is not limited to TIRF but applicable to many other systems involving multiple states of Brownian motion.