Three-Dimensional Imaging and Tracking for Constrained Brownian Motion of Single Nano-Scaled Particles in Solution

Objective-type total internal reflection fluorescence technique and Gaussian fit single particle tracking algorithm have been used to study the constrained Brownian motion characteristics of single fluorescent microspheres of 40 nm diameter in real-time. Three-dimensional tracking has been realized from the two-dimensional images by utilizing the exponential decay of the fluorescence intensity excited by the evanescent field. Trajectory analyses have shown that single fluorescent beads diffused in a constrained Brownian mode with a diffusion coefficient a little smaller than theoretical estimates in the plane-parallel direction.