Shapiro, Ian Ross McKay (2009) Observation of single-molecule rotational diﬀusion at microsecond timescales by polarized fluorescence correlation spectroscopy. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06052009-140716
This work presents a series of experimental and numerical studies of macromolecular organic, inorganic and biological structures, in all instances focusing on the behavior characteristic of individual discrete molecular elements. Chapters 1 and 2, beginning on pages 1 and 31, respectively, describe fabrication, use and numerical analysis of of single-walled carbon nanotube probes for amplitude-modulation atomic force microscopy. These studies reach the conclusion that the molecular structure and nanoscale surface interaction potential unique to carbon nanotubes collectively give rise to atomic force microscopy imaging artifacts manifesting as apparent lateral topographic resolution signiﬁcantly better than that predicted by the probe and sample structures. Chapter 3 (p. 61) presents a brief review of single-molecule microscopy, describes a generalized mathematical formalism for focusing polarized visible-spectrum electromagnetic radiation, and delineates the construction of a custom two-channel scanning confocal ﬂuorescence microscope system with single-photon detection capability for spectral- and polarization-resolved studies of individual mobile ﬂuorophores. This Chapter includes a theory-based optical analysis of the confocal probe volume structure and photoluminescence collection efficiency from 3D-polarized single-dipole emitters. The latter analysis was aided by introducing a modiﬁed Jones formalism using non-square matrix representation for polarization state changes in the speciﬁc context of confocal optics. Proper calculation of the expected confocal probe volume dimensions was essential for accurately interpreting experimental data in the following chapter. Additionally, the quantitative understanding that followed from analysis of 3D polarization state measurement by orthogonally polarized detection channels was critical to both the interpretation of experimental data and the numerical generation of simulated data in Chapter 5. Chapter 4 (p. 125) presents a generalized formalism for correlation analysis of the ﬂuorescence signal collected using the two-channel microscopy system described in Chapter 3. Particular focus was directed toward the theoretical auto- and cross-correlation traces anticipated from polarization-sensitive bivariate time series of photoluminescence emission from freely-rotating transition dipoles. Chapter 4 also presents population-resolved data collected from single F¨orster resonance energy transfer ﬂuorphore pairs conjugated to DNA oligomers as they undergo cleavage by restriction endonucleases. The endonuclease enzyme Michaelis constants KM measured for EcoRI and BglI via ﬂuorescence burst analysis were in agreement with prior literature. The success of these experiments provide concrete conﬁrmation of the microscope’s ﬂuorescence emission sensitivity and detection channel selectivity in the context of single-molecule experiments. Chapter 5 describes a polarized ﬂuorescence correlation spectroscopy (PFCS) investigation of liquid phase rotational diffusion by colloidal CdSe semiconductor nanocrystals possessing two-dimensional nondegenerate photoluminescence transition dipoles, as well as red ﬂuorescent protein (monomeric DsRed) and rhodamine-labeled phospholipids that possess more conventional one-dimensional ﬂuorescence transition dipoles. The experimental PFCS data collected from these samples is in close agreement with simulated PFCS data produced by a Monte Carlo rotational diffusion numerical routine that incorporates the microscope 3D polarization state sensitivity calculated in Chapter 3. Appendices beginning on page 221 include a matrix-based description of arbitrary 3D rotation that was used in the rotational diffusion simulations, Matlab code transcripts (p. 227), and an additional mathematical formalism based on information theoretic precepts (p. 242) for assessing directed causal relationships in bivariate time series data.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||atomic force microscopy; carbon nanotubes; confocal microscopy; fluorescence correlation spectroscopy; polarization; quantum dots; rotational diffusion; semiconductor nanocrystals; single-molecule fluorescence|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||2 June 2009|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||26 Jun 2009|
|Last Modified:||26 Dec 2012 02:51|
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