Biological Applications of Confocal Fluorescence Polarization Microscopy

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of metatetrahydroxyphenylchlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proofof- principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed

Complement factor B is critical for sub-RPE deposit formation in a model of DHRD/ML with features of age-related macular degeneration

EFEMP1 R345W is a protein misfolding-prone mutation causing Doyne honeycomb retinal dystrophy/Mallatia Leventinese (DHRD/ML), a rare blinding disease with similar clinical pathology to a common disease of age-related macular degeneration (AMD). Aged Efemp1R345W/R345W knock-in mice (Efemp1ki/ki mice) develop deposits on the basal side of retinal pigment epithelial cells, which is complement C3- dependent. We assessed alternative complement pathway component factor B (FB) in sub-RPE deposit formation in Efemp1ki/ki mice. RNA-seq analysis of Efemp1ki/ki mice comparing to Efemp1+/+ reveals increased unfolded protein response in posterior eye cups, decreased mitochondrial function in neural retina (both by 3-month-old), and increased inflammatory pathways in both tissues (at 17-month-old). Aged Efemp1ki/ki mice also exhibits elevated ocular complement protein activation with around two-fold (p<0.05) elevation of breakdown products iC3b and Ba by Western blot analysis. Proteomics analysis of eye lysate confirmed similar Efemp1 R345W-dysregulated pathways as detected by RNA-seq. Cfb deficiency partially normalizes these biological pathway changes in the eyes of female Efemp1ki/ki mice. Female Efemp1ki/ki mice dosed with a small molecule FB inhibitor from 10- to 12-month-old reduced sub-RPE deposits by 65% (p=0.051). Male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females and no elevation of ocular complement activation, therefore not affected by FB inhibitor. Sub-RPE deposits/drusen, seen in Efemp1ki/ki mice, are a common early clinical feature of DHRD/ML and AMD. The broader effect on Efemp1ki/ki mice by either Cfb deficiency or oral FB inhibition suggest that systemic inhibition of the alternative complement pathway is potentially an effective strategy for treatment of dry AMD and DHRD/ML