Calibration of optical tweezers for force microscopy

Abstract

Optical tweezers are an important tool in biophysics for their ability to noninvasively control microscopic particles, and measure forces in cellular environments. The trap must be calibrated for there to be a quantified force measurement. In this thesis, I use simulation to investigate the forces in optical tweezers and calibration of these forces. There are many methods which can be used to calibrate the forces. I first discuss the use of escape force calibration in detail. Using a combination of dynamic simulation and force calculations, I find that there is a zero axial force contour in the optical trap, which affects escape force measurements and explains behaviour which has been previously observed. I then use this escape force calibration for the calibration of the motility forces of isolated chromosomes, which provides information for the calculation of chromosome mitotic forces.I then introduce a new method of calibration which does not require any knowledge of the particle being optically trapped, or its environment. This is a method of absolute calibration. I require only the assumption that the force–position curve is monotonic, making no assumptions about the sphericity of the trapped particle or viscosity of the medium. Using dynamic simulation of an optically trapped particle, I find that the accuracy and tolerance of this new method of absolute calibration is comparable or an improvement on that of other calibration methods.I then use my absolute calibration method for the calibration of nonoptical forces. I first discuss the calibration of forces from surfaces, then forces from swimmers. Through dynamic simulations, I find that wall forces can be calibrated with the combination of my absolute calibration method and another position-only calibration method. I find that for an analogue of biological swimmers, swimming with uniform velocity, the calibration is straightforward and resembles that of the walls. For a more complex swimming model, the calibration is not as straightforward and other factors may need to be included.The final topic of my thesis is on the optimisation of a two-photon polymerised optically-driven micromachine. I specifically consider a paddlewheel which can be optically trapped and spun to create a shear force in a cellular environment. Through calculations of the forces acting on the paddles of the paddlewheel, I provide the optimal dimensions of the paddles and an estimate on the shear forces it can exert.