The lack of photobleaching, minimal sample heating, and high acquisition rates associated with coherent anti-Stokes Raman scattering (CARS) microscopy make it an attractive approach for the chemically specific in vivo imaging of dynamical processes. However, imaging capabilities are currently confined to classes of macromolecules as opposed to specific molecular targets. The use of cyano and deuterium functional group labels, which possess Raman modes in a spectral region devoid of endogenous cellular resonances, has the potential to surmount this limitation, enabling imaging with chemical and molecular specificity using CARS microscopy. Herein, cyano and deuterium vibrational modes have been incorporated into Raman and CARS contrast agents capable of mediating biomolecular modification. Application of this approach will be demonstrated using hepatitis C virus (HCV) RNA and two model protein systems with the end goal of investigating dynamical aspects of HCV molecular virology in real-time in vivo using CARS microscopy. The addition of exogenous CARS labels to a biomolecule can have serious structural and functional consequences that may lead to the expression of a phenotype dictated by the effects of the modification rather than the system under investigation. Therefore, the structural and functional consequences of introducing CARS labels needed to be investigated before applying cyano and deuterium modified HCV RNA and proteins to in vivo analysis using CARS microscopy.

Structure determination of membrane proteins by solution nuclear magnetic resonance spectroscopy require that they can be incorporated into detergent micelles in order to ensure that they are maintained in a folded, water-soluble state. However, detergent molecules produce micelle-protein complexes that pose unique challenges for solution NMR studies. To improve the spectroscopic properties of these complexes, it is possible to use specific isotope labeling strategies developed for the study of large water-soluble proteins by solution NMR. Specifically, the use of a highly deuterated sample that retains protons only in the Val, Leu and Ile (delta1) methyl groups has the potential to be used to determine structures of polytopic helical membrane proteins. However, while the quality of structure that can be obtained from this approach has been examined for water-soluble proteins, its utility for this class of membrane proteins has not been systematically investigated. This thesis evaluates the utility of this method for structure determination of membrane proteins by exploring structure calculation strategies from simulated NMR data sets from membrane proteins of known structure. Here I present results for a set of membrane proteins that consist of pairs or bundles of hydrophobic alpha-helices. I analyzed the impact of intra- and inter-helical NOEs on structure quality for samples specifically labeled with protons only at the methyl and amide positions In addition, I also explored the role of dihedral angles and residual dipolar couplings in an effort to find general trends to improve the accuracy of structures for polytopic a-helical membrane proteins. Based on the results of these calculations it appears that obtaining a uniform distribution of inter-helical NOES and accurately identifying transmembrane helices for dihedral angle restraints are the most important factors determining the accuracy of these simulated membrane protein structures. These results should help to guide future structure determinations for polytopic alpha-helical membrane proteins of unknown structure.

This thesis focuses on various carborane compounds and their uses for targeted cellular imaging. Cellular imaging agents have been developed for the field of medical diagnosis and treatment for some time. Many different processes have been traditionally applied in these fields, including fluorescence tagging, but these are lacking in differentiation from cellular background signals. Herein, carborane compounds have been developed for use as Raman reporters with signature absorption for BH vibrations, inside of the cell silence vibrational range. Carboranes have also been studied for the application of Boron Neutron Capture Therapy, a binary radiation therapy technique. Coupling these two ideas has lead to the formation of specific cellular targeting agents utilizing the unique BH vibrations for imaging, as well as the possible application of BNCT to the malignant tissue. This process has been developed with the aid of silver nanoparticles, which have been shown to enhance the Raman signal up to a factor of 1014 with Surface Enhanced Raman Scattering (SERS) techniques. Functionalized carborane compounds have been developed in order to study BH vibrations, and carborane-containing functionalized silver nanoparticles have been applied to target anti-EGFR antibodies for malignant tissue detection. Resulting SERS images confirmed selective tissue targeting with nanoparticle aggregate hot spots. Overlaying scanning electron microscope images with SERS BH vibrational intensity maps provided additional information on concentrations of cell surface receptors and identifying intercellular structures. Elaboration of the carborane Raman reporter resulted in two other carboranes with key functional groups, which could increase solubility, and have additional Raman handles for identification.

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