Development of a magnetic manipulator for microbiological and single molecule investigations

Forces play a critical role in a wide range of biological phenomena from single protein conformational dynamics, cell division, and mucociliary clearance, to name a few. The majority of existing instruments for microbiological force application can be divided into two categories; those that can apply relatively high forces through the use of a physical connection to a probe, and those that apply significantly smaller forces with a detached probe. Magnetic manipulators utilizing high fields and high field gradients have been able to reduce this gap in maximum applicable force, but the size of such devices has limited their use in applications where high force and high numerical aperture (NA) microscopy must be combined. This dissertation focuses on the development of a magnetic manipulation system that is capable of applying forces in excess of 700 pN on a 1 micron paramagnetic particle and 13 nN on a 4.5 micron paramagnetic particle, forces over the full 4pi steradians, and a bandwidth in excess of 3 kHz while remaining compatible with a commercially available high NA microscope objective. This device has been combined with a feedback enhanced, high resolution (2.4nm), high bandwidth (10 kHz), long range (100 micron xyz range) laser tracking system. The design and testing of this instrument is discussed, as well as biological investigations that take full advantage of the instrument's capabilities. This dissertation concludes with the presentation of a technique that utilizes polymer dynamics to determine tension and polymer specific properties in both in vitro and in vivo settings. Future investigations involving this analysis technique will benefit greatly from the application of the manipulation device described in this dissertation.