By applying the three-dimensional total internal reflection velocimetry (3D-TIRV) technique to freely suspended micron-sized fluorescent particles, we are able to simultaneously observe the three-dimensional anisotropic hindered diffusion for values of the gap-size-to-radius ratio much less than one. We demonstrate that the 3D-TIRV can be used to accurately track freely suspended 1.5-mu m radius particles. The displacement measurements reveal that the hindered diffusion coefficients are in close agreement with the theoretical values predicted by the asymptotic solutions of Brenner [Chem. Eng. Sci. 16, 242 (1961)] and Goldman [Chem. Eng. Sci. 22, 637 (1967)] for gap-size-to-radius ratio much less than one, while hindered diffusion anisotropicity is simultaneously observed in all data sets.
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