When trapping a dielectric bead with optical tweezers, light backscattered by the bead interferes in the back focal plane of the objective with light reflected by the microscope cover slip. This well contrasted interference pattern can be used to calibrate precisely the relative position of the bead with respect to the center of the trapping laser, as well as the stiffness of the trap. We compared four calibration methods previously implemented in forward detection, namely step response, Bayesian inference, power spectrum analysis and equipartition. We showed that they agree for different heights and trapping powers. Effect of acoustic noise on all methods was observed, and the step response method was shown to be the least sensitive. In addition to giving better access to the sample, this backscattered interference pattern provides precise location of the bead with respect to the laser, both laterally and axially, even when the bead is not trapped by the laser. We apply this to microrheology of blood clots, where the focused laser exerts a force on a bead confined by the fibrins of the clot but does not actually trap it .
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