Optimising optical tweezers for tracking and force measurement experiments

Optical Tweezers are a useful tool in many aspects of biology, including cell manipulation and microrheology. They are often used as piconewton force transducers, and are an effective tool for measuring forces acting upon optically trapped particle. To measure such forces, knowledge of the displacement of the particle from the trap centre is always needed. However, due to Brownian motion, a trapped particle is constantly moving and never at rest. In this case, one must track a bead over a set time, so as to gain an average displacement. In this thesis, we have improved and optimised this tracking procedure for biological samples in different ways. In Chapter 1 we discuss how Optical Tweezers work, how they are set up, and how we measure forces using them. In Chapter 2 we redesign a commercial Optical Tweezer Product to improve tracking data results. We also incorporate fluorescence imaging using a compact, low cost, LED illumination source. In Chapter 3 we combine fluorescence microscopy with state of the art Scientific cameras, to increase tracking frame rates and potentially improve our tracking data of fluorescent stained cells. This was part of a collaboration, where I helped to build the setup, took the data (using programs produced by one of my collaborators), and was part of the team to analyse it. In Chapter 4, we look at Low Reynolds number environments and discuss the benefits of viscous forces, and how it may be possible to make non-invasive, less harmful traps for biological samples. Again, this was part of a collaboration, where I was in charge of the experimental part. Here, I built in the static tweezer trap into a tweezer system, took position data and analysed it. A collaborator took control of analysing velocity data. Finally, in Chapter 5, we measure the accuracy of tracking in three dimensions using a stereomicroscope, by placing a Spatial Light Modulator (SLM) at the Fourier plane in the imaging arm. Again, this was a collaboration. I designed and manufactured the illumination head, helped design an acquisition program, and took the data. We discuss how all of these could optimise and advance the tracking of optically trapped particles, especially biological samples. Despite the obvious applications in biology, to allow a fair evaluation of the different tracking techniques, all of our experiments used samples of spherical beads, as they have known specifications, including fluorescence excitation and emission wavelengths, size, and amount of fluorophore stain