Fluorescence correlation spectroscopy and single molecule burst analysis were used to measure the effects of glass surface interactions on the diffusion of two common fluorescent dyes, one cationic and one anionic. The effects of dye–surface interactions on measured diffusion rates as a function of distance from the surface were investigated. Use of a three-axis piezo stage, combined with reference calibration measurements, enabled the accurate acquisition of surface-distance-dependent transport data. This analysis reveals attractive interactions between the cationic dye and the surface, which significantly alter the extracted diffusion values and persist in the measurements up to 1.0 μm from the surface. The Coulomb attraction between the cationic dye and the surface also results in rare, long-lived association events that lead to irreproducibility in extracted diffusion values. In addition to an assignment of the association lifetime for these events, this paper demonstrates that, if experiments must be performed with cationic probes near a glass surface, the use of solution electrolytes can eliminate deleterious dye–surface interactions, as the dyes were tested in a variety of environments. Finally, our data demonstrate that a better dye choice is an anionic probe, which exhibits no depth dependence of diffusion characteristics above a glass surface.
展开▼
