Optical and magnetic tweezers for applications in single-molecule biophysics and nanotechnology

Resumen

Over the last few decades, continuous improvements of microscopy techniques have constituted one of the driving forces for the interdisciplinary field of single-molecule biophysics -- a scientific environment that bridges areas of physics, biology, chemistry and micro-/nanotechnology, amongst others. This dissertation deals with the design, construction and use of two types of instruments based upon optical microscopes: optical and magnetic tweezers to manipulate individual biomolecules such as DNA or proteins in solution. First of all, the assembly of a tailor-made optical tweezers setup in an inverted microscope configuration with a single objective is discussed. Such a design provides free access to the top side of the sample stage, facilitating for instance the implementation of different flow cell types. Thanks to the development of an application-specific software, the system can exploit both video- and laser-based position detection methods -- the latter via back-reflections of an additional low-power laser. We have calibrated the trap stiffness for polystyrene microspheres by spectral analysis of thermal fluctuations and viscous drag measurements. Using a micropipette as a mechanical anchor point for pulling experiments, trapping-laser-induced thermal drift effects can be minimised by active control of the objective temperature and sample stage position corrections. In its current state, the entire assembly is able to carry out and measure force--extension curves of various DNA substrates, with a precision that resembles the one of other optical trapping instruments. In a second project, an optical tweezers device equivalent to the aforementioned is employed in combination with an ionic current sensing platform to probe electrically induced fluid flows through glass nanopipettes. Rotation rates and frictional forces, exhibited by optically trapped microspheres with a small cavity, relate directly to the local flow field outside the pore and comply with the Landau--Squire solution of the Navier--Stokes equations. Raster-scanning the area in front of the pipette tip with this micrometric anemometer, the volume flow rate at the pore exit turns out to be on the order of tens of picolitres per second -- below the majority of velocities assessable with other flow-measuring techniques. Finally, the implementation of a customised temperature control system in a magnetic tweezers instrument is presented. Simplicity and flexibility of the thermostat make it attractive for other techniques based on inverted microscopes. Between ambient and physiological conditions, thermal settings inside the buffer volume are adjustable with a precision of 0.1ºC. We have tested the complete setup with the molecular motor AddAB, a helicase--nuclease protein complex that moves along double-stranded DNA. In comparison with results from bulk assays, the thermally stabilised magnetic tweezers yield the same relative exponential increase of AddAB velocity with temperature. Absolute translocation rate values from single-molecule and ensemble measurements can be matched by saturating the effective ATP concentration near the protein in the magnetic tweezers flow cell.