Total internal reflection fluorescence microscopy for characterizing biochemical interactions

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  • March 21, 2019
Creator
  • Steele, Bridgett L.
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Total internal reflection fluorescence (TIRF) microscopy is a widely used technique for the study of biologically relevant processes both in vitro and in vivo. In TIRF, an incident light beam propagates through a transparent solid and encounters a solid/liquid interface at an angle sufficient enough such that the light undergoes total internal reflection, which generates an evanescent field that decays exponentially with distance from the surface. Thus, the main advantage of TIRF is that it selectively excites fluorescent molecules at or near the solid-solution interface. TIRF is used to study the thermodynamic and kinetic properties of three systems in this dissertation. Pregnane X receptor (PXR) is a member of the nuclear receptor family of ligand activated transcriptional factors that regulate gene expression. Using TIRF, the ligand-dependent binding of PXR to the steroid receptor coactivator-1 (SRC-1) is studied in the presence of the known PXR activator, rifampicin. In this system, the ligand binding domain of PXR is immobilized at the solid-solution interface, and fluorescently-labeled SRC-1 peptide in solution is used to mimic the full length SRC receptor. By measuring the fluorescence intensity as a function of the solution concentration of fluorescently-labeled SRC-1 at constant, increasing concentrations of rifampicin, four equilibrium binding constants are obtained, which illustrate the cyclic nature of these interactions. Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens in the extracellular matrix. Upon activation through collagen binding, DDR2 regulates cell adhesion, proliferation, and extracellular matrix remodeling. To study the binding of the extracellular domain of DDR2 to collagen, methods to deposit thin films of aligned or unaligned collagen I on fused silica slides were developed. Additionally, to characterize the effect of oligomerization of DDR2 on collagen binding, methods to separate monomeric, dimeric, and aggregates of DDR2 were also explored. This work represents the preliminary steps necessary to study the binding of surface immobilized collagen to fluorescently-labeled DDR2 using TIRF. A new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching (TIR-CP) is presented. In this method, small structures, such as prokaryotic cells or isolated eukaryotic organelles, containing fluorescent molecules are adhered to a surface. This surface is continuously illuminated by an evanescent wave created by total internal reflection. The characteristic length describing the decay of the evanescent intensity with distance from the surface is smaller than the structures. The fluorescence decay rate resulting from continuous evanescent illumination is monitored as a function of the excitation intensity. The data at higher excitation intensities provide apparent translational diffusion coefficients for the fluorescent molecules within the structures because the decay results from two competing processes (the intrinsic photobleaching propensity and diffusion in the small structures). The theoretical basis for the technique is presented and the applicability of the technique is demonstrated by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of green fluorescent protein (GFP) in Escherichia coli cells.
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  • In Copyright
Advisor
  • Thompson, Nancy
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
Date uploaded
  • October 11, 2010

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