Motion of single DNA molecules at a liquid-solid interface as revealed by variable-angle evanescent-field microscopy.

Area of Science:

• Biophysics
• Surface Science
• Microscopy

Background:

• Studying single molecule dynamics at solid/liquid interfaces is crucial for understanding surface interactions.
• Existing microscopy techniques have limitations in resolving molecular motion near surfaces.

Purpose of the Study:

• To develop and validate a variable-angle total-internal-reflection fluorescence microscope (VATIRFM) for imaging single DNA molecule dynamics.
• To analyze the influence of molecular diffusion and evanescent-field layer (EFL) thickness on experimental counts.
• To elucidate the distance-dependent dynamics of molecules near surfaces.

Main Methods:

• Construction of a VATIRFM with a wide range of incident angles.
• Development of an ImageJ-based algorithm for single-molecule counting.
• Application of a one-dimensional random-walk diffusion model for simulation and analysis.
• Experimental validation of the model with DNA molecule dynamics.

Main Results:

• Experimental counts are significantly affected by molecular diffusion and EFL thickness.
• A simulation model accurately fits experimental counting data.
• Measured fluorescence intensities do not directly correlate with molecular distances from the surface.
• Counting results reflect distance-dependent molecular dynamics rather than concentration within the EFL.

Conclusions:

• VATIRFM is a powerful tool for studying molecular repulsion/attraction within hundreds of nanometers of a surface.
• Molecules exhibit electrostatic repulsion at distances beyond the electrical double layer at low ionic strengths.
• The technique provides detailed insights into single-molecule motion and surface interactions.