Accurate Calibration of Optical Tweezers close to a glass surface using interference rings in backscattered light

Detecting the retro-reflected trap beam is a convenient way to measure forces applied by optical tweezers on biological systems, leaving free top access to the sample and making it easier to combine with fluorescence and phase contrast imaging. Doing so in the back focal plane of the microscope objective makes the calibration insensitive to the joint displacement of the trap and bead. Here, in addition, we take advantage of the very well contrasted interference rings observed in this specific plane to extract with good accuracy the height of the trapped bead. We thus map the viscous drag dependence close to the surface and reach an agreement between four calibration techniques, on an accurate value of the trap stiffness down to 2 micron distance to the surface and up to 600 mW of laser power. Combining this detection scheme with phase contrast microscopy, we show that the phase ring in the back focal plane of the objective overlaps with the interference rings and must be deported in a conjugate plane on the imaging path.