A variable-angle total-internal-reflection fluorescence microscope (VATIRFM) capable of providing a large range of incident angles was constructed for imaging single DNA molecule dynamics at a solid/liquid interface. An algorithm using a public-domain image-processing program, ImageJ, was developed for single-molecule counting. The experimental counts at various incident angles with different evanescent-field layer (EFL) thicknesses are affected by molecular diffusion. The dynamics of molecules near the surface and the observed counts in the VATIRFM are elucidated using a limited one-dimensional random-walk diffusion model. The simulation fits well with the experimental counting results. Further analysis using the simulation reveals the details of single-molecule motion. One implication is that the measured intensities cannot be used directly to determine the distances of molecules from the surface, though the majority of fluorescence does come from the EFL. Another implication is that rather than providing molecular concentrations within EFL the experimental counting results depict the distance-dependent dynamics of molecules near the surface. Thus, the VATIRFM could be a powerful technique to study the surface repulsion/attraction of molecules within a few hundred nanometers of the surface. Further studies show that molecules at low ionic strengths experience electrostatic repulsion at distances much further away from the surface than the calculated thickness of the electrical double layer.
Scopus Subject Areas
- Physical and Theoretical Chemistry
- Surfaces, Coatings and Films
- Materials Chemistry