A novel three-dimensional tracking technique of nanoparticles in nanometer spatial resolution using a Ratiometric Total Internal Reflection Fluorescence Microscopy (R-TIRFM) is presented. Evanescent waves from the total internal reflection of an argon-ion laser (488 nm) are used to generate a thin sliced illumination field with its effective visualization range of 544-nm, equivalent to twice of the penetration depth of the evanescent wave field. Fluorescence-coated polystyrene spheres of 500-nm diameter (SG = 1.05) are used as tracers subjected to the Brownian diffusive motions. A ratiometric analysis of the fluorescence particle images together with a neural network particle-pair identification algorithm is used to track the tracer particle locations across multiple image frames in full three-dimensional ways. The techniques are used to examine the Brownian diffusive motion of nanoparticles as they approach the very near-wall region within a few hundred nanometers of a glass-water interface. The experimental results show that the measured Brownian diffusion coefficient is in good agreement with the theoretical hindered diffusion coefficient near a wall.

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