Development of instrumentation for sum frequency spectral imaging combined with confocal fluorescence imaging and additional topics

Understanding surfaces and interfacial organization in material and biological systems is critical across nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is thus necessary to address new questions and further current investigations. Subsequently, surface characterization is of great interest. In this work I present the design and construction of a novel microscope that not only visualize surfaces with high resolution confocal imaging but also provide unique label free lateral organization information through sum frequency microscopy. Sum frequency is a surface specific spectroscopy that provides organizational and vibrational information. The instrument detailed in this work takes advantage of sum frequency’s inherent surface specificity for surface visualization. Combined with confocal microscopy the detailed instrument allows surfaces to be visualized using two independent methods. Sum frequency microscopy is show to elucidate surface defects that would have been obscured in a traditional application. Two additional efforts are presented. First, characterization of a cushioned phospholipid bilayer’s main phase transition temperature and mobility is measured using z-scan fluorescence correlation spectroscopy. Three supporting surfaces are studied: glass, a hydrated polymer cushion, and an actin monolayer. Second, a novel Raman active nanoprobe is compared to a popular fluorophore in the context of confocal microscopy. The work I present in this thesis seeks to develop surface specific and visualization techniques applicable to a wide range of systems.