Optical tweezers combined with interference reflection microscopy for quantitative trapping and 3D imaging

Optical tweezers are an indispensable tool in biophysical single-molecule studies. They provide the ability to mechanically probe the characteristics of biological processes, such as active transport of cargo by molecular motors. To this end, functionalized (sub)micronsized dielectric particles are held in a tightly focused laser trap while external forces lead to displacements of the particle from the trap center. The measurement and calibration of these displacements yield insights into the mechanical properties of the molecule of interest. The study of molecular motors, such as kinesins, is carried out in in vitro surface-based experimental assays. The experimental needs for such assays are challenging. The instrument must be stabilized, i.e. decoupled form external noise, and drift must be minimized, and it needs to be combined with state of the art microscopy techniques to visualize the sample. These are, on the one hand, single-molecule fluorescence detection and, on the other hand, robust label-free imaging of diffraction limited specimen. The latter is commonly realized by differential interference contrast (DIC) microscopy, which is an expensive and rather complicated technique that also restricts the design of the optical tweezers and, therefore, reduces the experimental possibilities. Optical tweezers experiments, moreover, rely on precise and reliable calibration. Despite its importance, calibration is, at times, carried out with obsolete methods or based on vague assumptions. Especially, in the vicinity of the sample surface, where hydrodynamic effects can have a significant influence, such assumptions fail largely. Here, height-dependent active power spectral density analysis of the Brownian motion of the trapped particle can ameliorate these inaccuracies, but—compared to other methods—is rather cumbersome, timeconsuming and easy-to-use solutions are lacking. In this work I designed and assembled an optical tweezers setup combined with total internal reflection fluorescence (TIRF) microscopy. Furthermore, I succeeded to reduce design restrictions of the optical tweezers by combining it with interference reflection microscopy, which is a simple, cost-efficient and robust contrast technique that can visualize diffraction limited specimen in three dimensions, such as microtubules. Moreover, I was able to use this technique to determine the three-dimensional profile of an upward bent microtubule which I used to simultaneously calibrate the evanescent field depth of the TIRF microscope. In addition, I programmed a free and open-source optical tweezers calibration software, PyOTC, that provides the means for height-dependent active power spectral density analysis. My work will possibly influence the design of optical tweezers instruments for surface-based experiments. LED-based IRM could further improve or complement label-free detection techniques such as interferometric scattering microscopy. The free and open-source calibration software package could help to precisely calibrate optical tweezers data. Moreover, because the source is available to anybody, calibration and therefore the analysis of optical tweezers data will be more transparent to the scientific community.