Near-wall hindered Brownian diffusion of nanoparticles examined by three-dimensional ratiometric total internal reflection fluorescence microscopy (3-D R-TIRFM)

A three-dimensional nanoparticle tracking technique using ratiometric total internal reflection fluorescence microscopy (R-TIRFM) is presented to experimentally examine the classic theory on the near-wall hindered Brownian diffusive motion. An evanescent wave field from the total internal reflection of a 488-nm bandwidth argon-ion laser is used to provide a thin illumination field on the order of a few hundred nanometers from the wall. Fluorescence-coated polystyrene spheres of 200 +/- 20 nm diameter (specific gravity=1.05) are used as tracers and a novel ratiometric analysis of their images allows the determination of fully three-dimensional particle locations and velocities. The experimental results show good agreement with the lateral hindrance theory, but show discrepancies from the normal hindrance theory. It is conjectured that the discrepancies can be attributed to the additional hindering effects, including electrostatic and electro-osmotic interactions between the negatively charged tracer particles and the glass surface.