[
    {
        "id": "thesis:5270",
        "collection": "thesis",
        "collection_id": "5270",
        "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06262009-144008",
        "primary_object_url": {
            "basename": "thesis.pdf",
            "content": "final",
            "filesize": 4996181,
            "license": "other",
            "mime_type": "application/pdf",
            "url": "/5270/11/thesis.pdf",
            "version": "v6.0.0"
        },
        "type": "thesis",
        "title": "Biochemical Reactions in Confined Space",
        "author": [
            {
                "family_name": "Liu",
                "given_name": "Yu",
                "clpid": "Liu-Yu"
            }
        ],
        "thesis_advisor": [
            {
                "family_name": "Collier",
                "given_name": "C. Patrick",
                "orcid": "0000-0002-8198-793X",
                "clpid": "Collier-C-P"
            }
        ],
        "thesis_committee": [
            {
                "family_name": "Goddard",
                "given_name": "William A., III",
                "orcid": "0000-0003-0097-5716",
                "clpid": "Goddard-W-A-III"
            },
            {
                "family_name": "Collier",
                "given_name": "C. Patrick",
                "orcid": "0000-0002-8198-793X",
                "clpid": "Collier-C-P"
            },
            {
                "family_name": "Beauchamp",
                "given_name": "Jesse L.",
                "orcid": "0000-0001-8839-4822",
                "clpid": "Beauchamp-J-L"
            },
            {
                "family_name": "Phillips",
                "given_name": "Robert B.",
                "orcid": "0000-0003-3082-2809",
                "clpid": "Phillips-R"
            }
        ],
        "local_group": [
            {
                "literal": "div_chem"
            }
        ],
        "abstract": "The understanding of biological systems relies on the accurate description of the interaction among biomolecules. This knowledge can be obtained by in vitro assays involving interacting partners with well-defined compositional, spatial, and temporal constraints. The distinguishing features of living systems, namely, low copy number, crowded environment, and spatial compartmentalization, are usually absent in most in vitro experiments reported in the literature. This thesis discusses the implications of low copy number and spatial constraints using theoretical and computational methods in some model systems. Furthermore, two experimental platforms, based on the recent development of microfluidic techniques, are described in detail. In the first implementation, micronsized chambers fabricated using soft lithography provide a high-throughput reactor array whose size and composition can be configured to mimic the in vivo environment. The second design reports the generation and manipulation of femtoliter-volume water-in-oil droplets. A model biochemical reaction catalyzed by beta-galactosidase is observed in both reactors with precisely defined initiation time, opening the way to monitor transient kinetics in addition to steady-state behavior. Additionally, the enzymatic activity exhibits a negative correlation with the size of water-in-oil droplets when the nominal concentrations of reagents are kept the same. This surprising result is analyzed in detail by carefully designed control experiments, and attributed to the shear-induced redistribution of surfactant employed to stabilize the water/oil interface. Specifically, smaller droplets experience bigger shear stress, which change the surface concentration of surfactant and allow for the nonspecific binding of proteins to the interface. Surface-bound enzymes are denatured, leading to reduced catalytic activity. This highly dynamic process is hardly detectable by other methods such as tensiometry or direct fluorescence imaging of the interface.",
        "doi": "10.7907/W9H8-8T44",
        "publication_date": "2010",
        "thesis_type": "phd",
        "thesis_year": "2010"
    },
    {
        "id": "thesis:2467",
        "collection": "thesis",
        "collection_id": "2467",
        "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06052009-140716",
        "primary_object_url": {
            "basename": "Ian_Shapiro_PhD_thesis.pdf",
            "content": "final",
            "filesize": 10319534,
            "license": "other",
            "mime_type": "application/pdf",
            "url": "/2467/1/Ian_Shapiro_PhD_thesis.pdf",
            "version": "v2.0.0"
        },
        "type": "thesis",
        "title": "Observation of Single-Molecule Rotational Diffusion at Microsecond Timescale by Polarized Fluorescence Correlation Spectroscopy",
        "author": [
            {
                "family_name": "Shapiro",
                "given_name": "Ian Ross McKay",
                "clpid": "Shapiro-Ian-Ross-McKay"
            }
        ],
        "thesis_advisor": [
            {
                "family_name": "Collier",
                "given_name": "C. Patrick",
                "clpid": "Collier-C-P"
            }
        ],
        "thesis_committee": [
            {
                "family_name": "Okumura",
                "given_name": "Mitchio",
                "clpid": "Okumura-M"
            },
            {
                "family_name": "Collier",
                "given_name": "C. Patrick",
                "clpid": "Collier-C-P"
            },
            {
                "family_name": "Beauchamp",
                "given_name": "Jesse L.",
                "clpid": "Beauchamp-J-L"
            },
            {
                "family_name": "Goddard",
                "given_name": "William A., III",
                "clpid": "Goddard-W-A-III"
            }
        ],
        "local_group": [
            {
                "literal": "div_chem"
            }
        ],
        "abstract": "<p>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 signi\ufb01cantly better than that predicted by the probe and sample structures.</p>\r\n\r\n<p>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 \ufb02uorescence microscope system with single-photon detection capability for spectral- and polarization-resolved studies of individual mobile \ufb02uorophores. 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 modi\ufb01ed Jones formalism using non-square matrix representation for polarization state changes in the speci\ufb01c 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.</p>\r\n\r\n<p>Chapter 4 (p. 125) presents a generalized formalism for correlation analysis of the \ufb02uorescence 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\u00a8orster resonance energy transfer \ufb02uorphore pairs conjugated to DNA oligomers as they undergo cleavage by restriction endonucleases. The endonuclease enzyme Michaelis constants KM measured for EcoRI and BglI via \ufb02uorescence burst analysis were in agreement with prior literature. The success of these experiments provide concrete con\ufb01rmation of the microscope\u2019s \ufb02uorescence emission sensitivity and detection channel selectivity in the context of single-molecule experiments.</p>\r\n\r\n<p>Chapter 5 describes a polarized \ufb02uorescence 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 \ufb02uorescent protein (monomeric DsRed) and rhodamine-labeled phospholipids that possess more conventional one-dimensional \ufb02uorescence 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.</p>\r\n\r\n<p>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.</p>\r\n",
        "doi": "10.7907/KVDR-9429",
        "publication_date": "2009",
        "thesis_type": "phd",
        "thesis_year": "2009"
    }
]