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Observation of Single-Molecule Rotational Diffusion at Microsecond Timescale by Polarized Fluorescence Correlation Spectroscopy


Shapiro, Ian Ross McKay (2009) Observation of Single-Molecule Rotational Diffusion at Microsecond Timescale by Polarized Fluorescence Correlation Spectroscopy. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/KVDR-9429.


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
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Collier, C. Patrick
Thesis Committee:
  • Okumura, Mitchio (chair)
  • Collier, C. Patrick (co-chair)
  • Beauchamp, Jesse L.
  • Goddard, William A., III
Defense Date:2 June 2009
Record Number:CaltechETD:etd-06052009-140716
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2467
Deposited By: Imported from ETD-db
Deposited On:26 Jun 2009
Last Modified:26 Nov 2019 20:37

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