Total Internal Reflection Microscopy

Total internal reflection microscopy (TIRM) can measure the interaction potentials between a colloidal particle and a wall with femtonewton resolution. The equilibrium distribution of the particle-wall separation distance z is sampled monitoring the intensity I scattered by the Brownian particle under evanescent illumination.

From this one can determine the distance resolved interaction potential and corresponding forces with femtonewton resolution. The central point of the data analysis is the a priori knowledge of the relation between the measured scattering intensity I and the corresponding particle distance z. For short penetration depths of the evanescent field, it has been demonstrated that I(z) ∝ exp(−z/d). This, however, poses considerable constraints to the experimental conditions and the range of forces where TIRM can be applied. We introduced a method to experimentally determine I(z) by making solely use of the distance-dependent hydrodynamic interactions between the particle and the wall. We demonstrate, that our method largely extends the range of conditions accessible with TIRM, and even allows measurements on highly reflecting gold surfaces where multiple reflections lead to large deviations from an exponential I(z) relationship.