Killian, Jessica Lynn2017-04-042019-02-012017-01-30Killian_cornellgrad_0058F_10133http://dissertations.umi.com/cornellgrad:10133bibid: 9906023https://hdl.handle.net/1813/47776Optical trapping is a powerful and sensitive technique that enables the direct measurement and manipulation of biological samples on the piconewton and nanometer scales. In examining the evolution and maturation of optical trapping instrumentation in the field of single-molecule biophysics, two main thrusts become apparent; a subset of instruments have aimed to push the limits of resolution, seeking to capture the finest details of biomechanical processes, while a second subset of instruments have explored the integration of optical trapping with other measurement techniques, with the goal of creating multidimensional, hybrid instruments. Unfortunately, many hybrid trapping instruments are forced to sacrifice measurement resolution, or relinquish their measurement capabilities entirely, when the combination of techniques cannot accommodate the stringent demands of traditional optical trapping detection methods. Thus, the full potential of hybrid optical trapping instruments has remained elusive. We have taken on this challenge by developing a highly-versatile optical tweezers instrument that can achieve sub-nanometer position resolution without the use of a second microscope objective, making high-resolution optical trapping measurements uniquely compatible with a broad array of bulky and/or opaque sample-side devices. Our instrument consists of a time-shared, dual optical trap implemented in an entirely custom-built, free-standing fluorescence microscope, with camera-based position detection. We have developed a robust and accurate set of tracking techniques that allow us to make the first direct measurements of relative bead displacement within an optical trap with sub-nanometer resolution using image tracking. These capabilities pave the way for the next generation of optical tweezer hybrids, which we envision as versatile tools that can integrate multiple measurement modalities and complex substrates without sacrificing resolution, to expand the breadth and clarity of our window into the microscopic and sub-microscopic biological worlds.en-USBiophysicsPhysicsDual TrapImage TrackingOptical TrapOptical TweezersResolutionSingle Moleculedissertation or thesishttps://doi.org/10.7298/X4R49NS2