Measurement of Colloidal Forces with TIRM

During the last ten years, we have developed a new experimental technique called Total Internal Reflection Microscopy. Using TIRM we can monitor the separation distance between a single microscopic sphere immersed in an aqueous solution and a transparent plate. Because the distance is calculated from the intensity of light scattered by the sphere (3 to 30 microns in diameter) when illuminated by an evanescent wave, this technique provides a sensitive, nonintrusive, and instantaneous measure of the distance between the sphere and the plate. Changes in distance as small as 1 nm can be detected. From the equilibrium distribution of separation distances sampled by Brownian motion, we determine the potential energy profile in the vicinity of the minimum formed by gravitational attraction and double-layer repulsion or steric repulsion caused by adsorbed soluble polymer. Forces as small as 0.01 piconewtons can be detected. We have also measured van der Waals attraction, the radiation pressure exerted by a focussed laser beam, receptor-mediated interaction between antigen and antibodies, and steric repulsion due to adsorbed polymer layers. From the autocorrelation of the temporal fluctuations in scattering intensity, we have inferred the value of the normal component of the diffusion coefficient, which is about two orders of magnitude smaller than the bulk value owing to the close proximity of the sphere to the wall. This provides the first experimental test of Einstein's equation (relating mobility and diffusion coefficient) in a colloidal force field.