Objective-type dark-field illumination for scattering from microbeads

We introduce a method for detecting and tracking small particles in a solut ion near a surface. The method is based on blocking the backreflected illum ination beam in an objective-type total internal reflection microscope, lea ving unhindered the light scattered by the particles and resulting in dark- field illumination. Using this method, we tracked the motion of 60-nm polys tyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold part icles with a signal-to-noise ratio of 5. We illustrate the method's use by following the Brownian motion of small beads attached by short DNA tethers to a substrate. (C) 2001 Optical Society of America.