Hindered diffusion of colloidal particles very near to a wall: Revisited

Total internal reflection microscopy is a technique for monitoring changes in the distance between a single microscopic sphere and a flat plate by mea suring the intensity of light scattered by the sphere when illuminated by a n evanescent wave. A histogram of scattering intensities can be used to con struct the potential energy profile as a function of distance relative to t he most probable distance. Thus potential energies can be measured to withi n a fraction of kT while changes in distance can be measured to within 1 nm . An autocorrelation of the scattering intensities can be used to deduce an average diffusion coefficient of the sphere, which is found to be only a f ew percent of the Stokes-Einstein value, owing to the close proximity of th e plate. The analysis of the intensity-autocorrelation function presented h ere can be used to deduce an absolute value for the most probable separatio n distance, without a priori knowledge of the functional form of the PE pro file and in the presence of a constant background scattering intensity. Thi s "hydrodynamic" separation distance is found to be within a few percent of the "optical" separation distance found independently by comparing the int ensity at the most probable distance with the intensity of the same particl e in contact with the plate. Since the particle does not need to be brought into contact with the plate, the hydrodynamic method is well suited for de termining the absolute separation distance with deformable particles like l iquid droplets, vesicles or biological cells. Moreover, the hydrodynamic se paration can be immediately calculated without any additional experiments. However, accurate determination of the hydrodynamic separation requires an accurate value for the particle size, which must be determined independentl y. (C) 2000 American Institute of Physics. [S0021-9606(00)70127-9].