Accurate calibration of optical tweezers close to a glass surface using interference rings in backscattered light

Mechanical forces play an important role in the behaviour of cells, from differentiation to migration and the development of diseases. Optical tweezers provide a quantitative tool to study these forces and must be combined with other tools, such as phase contrast and fluorescence microscopy. Detecting the retro-reflected trap beam is a convenient way to monitor the force applied by optical tweezers, while freeing top access to the sample. Accurate in situ calibration is required especially for single cells close to a surface where viscosity varies rapidly with height. Here, we take advantage of the well contrasted interference rings in the back focal plane of the objective to find the height of a trapped bead above a cover slip. We thus map the viscous drag dependence close to the surface and find agreement between four different measurement techniques for the trap stiffness down to 2 mu m above the surface. Combining this detection scheme with phase contrast microscopy, we show that the phase ring in the back focal plane of the objective must be deported in a conjugate plane on the imaging path. This simplifies implementation of optical tweezers in combination with other techniques for biomechanical studies.