In optical trapping, accurate determination of forces requires calibration of the position sensitivity relating displacements to the detector readout via the V-nm conversion factor (β). Inaccuracies in measured trap stiffness (k) and dependent calculations of forces and material properties occur if β is assumed to be constant in optically heterogeneous materials such as tissue, necessitating calibration at each probe. For solid-like samples in which probes are securely positioned, calibration can be achieved by moving the sample with a nanopositioning stage and stepping the probe through the detection beam. However, this method may be applied to samples only under select circumstances. Here, we introduce a simple method to find β in any material by steering the detection laser beam while the probe is trapped. We demonstrate the approach in the yolk of living Danio rerio (zebrafish) embryos and measure the viscoelastic properties over an order of magnitude of stress-strain amplitude.
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