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Fluorescence-force spectroscopy at the single molecule level

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Title: Fluorescence-force spectroscopy at the single molecule level
Author(s): Zhou, Ruobo
Director of Research: Ha, Taekjip
Doctoral Committee Chair(s): Aksimentiev, Aleksei
Doctoral Committee Member(s): Ha, Taekjip; Selvin, Paul; Stack, John
Department / Program: Physics
Discipline: Physics
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): single molecule detection fluorescence microscopy optical tweezers
Abstract: During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. As the single molecule techniques become mature, there is increasingly pressing need to maximize the information content of the analysis in order to be able to study more complex systems that better approximate in-vivo conditions. Here, we develop a fluorescence-force spectroscopy method to combine single-molecule fluorescence spectroscopy with optical tweezers. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like single molecule Förster (or fluorescence) resonance energy transfer (FRET) whose detectable distance range is approximately 3-10 nm, we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments. We demonstrate the applications of this method to various biological systems including: 1) to measure the effect of very low forces on the nanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2) to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) to calibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding of single proteins. The results could not have been obtained with fluorescence or force measurement alone, and clearly demonstrates the power and generality of our approach. Finally, we show that self-quenching of two identical fluorophores can be used to detect small conformational dynamics corresponding to sub-nanometer distance changes of single molecules in a FRET-insensitive short range (< 3 nm), extending the detectable distance range of our fluorescence-force spectroscopy method.
Issue Date: 2012-06-27
URI: http://hdl.handle.net/2142/31921
Rights Information: Copyright 2012 Ruobo Zhou
Date Available in IDEALS: 2012-06-27
Date Deposited: 2012-05
 

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