McHale, Kevin L. (2008) Feedback tracking and correlation spectroscopy of fluorescent nanoparticles and biomolecules. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05072008-204627
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The best way to study dynamic fluctuations in single molecules or nanoparticles is to look at only one particle at a time, and to look for as long as possible. Brownian motion makes this difficult, as molecules move along random trajectories that carry them out of any fixed field of view. We developed an instrument that tracks the Brownian motion of single fluorescent molecules in three dimensions and in real-time while measuring fluorescence with nanosecond time resolution and single-photon sensitivity. The apparatus increases observation times by approximately three orders of magnitude while improving data-collecting efficiency by locking tracked objects to a high-intensity region of the excitation laser.
As a first application of our technique, we tracked and studied the fluorescence statistics of semiconductor quantum dots. Our measurements were well resolved at 10ns correlation times, allowing measurement of photon anti-bunching on single particles in solution for the first time. We observed variations of (34 [...] 16)% in the fluorescence lifetimes and (23 [...] 18)% in the absorption cross-sections within an aqueous quantum dot sample, confirming that these variations are real, not artifacts of the immobilization methods previously used to study them. Additionally, we studied quantum dot fluorescence intermittency and its dependence on 2-mercaptoethanol, finding evidence that the chemical suppresses blinking on short time-scales (< 1s) by reducing the lifetime of the dark state.
Finally, we studied the translational and intramolecular Brownian motion of [lambda]-phage DNA molecules. Our apparatus decouples these motions almost completely, and yielded a translational diffusion coefficient estimate D=(0.71 [...] 0.05)[...] lying between previous measurements for this molecule under identical solution conditions but with less precise techniques. Our measurements show clear evidence of intramolecular motion of the polymer chain in the form of statistical correlations on time-scales up to 1s, but we have not yet been able to determine the influence of solvent interactions on these dynamics.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA; FCS; polymer; quantum dot; Rouse; Zimm|
|Degree Grantor:||California Institute of Technology|
|Division:||Engineering and Applied Science|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||8 January 2008|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||20 May 2008|
|Last Modified:||10 Dec 2014 20:21|
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