Looking at the interface: Novel Microscopy Techniques for Quantitative Phase Imaging in Total Internal Reflection

Abstract

Looking at the interfaces is very important for many biological and chemical applications: from the imaging of thin polymeric films to the study of cell/substrate interactions. Total Internal Reflection (TIR) microscopy and Surface Plasmon Resonance imaging (SPRi) are usually the preferred techniques when high sensitivity of measurement is required. They use super critical illumination to create evanescent waves at the sample/substrate boundary. The very short range of penetration of these evanescent waves allows to retrieve information about few hundreds of nanometers from the contact surface. Evanescent wave microscopy has proven itself as a good tool for the characterization of thin films, cellular focal adhesions and biomolecular binding events. However, despite the high sensitivities achieved so far, each technique alone has a limited detection range with optimal sensitivity. The main goal of this work is to show how these techniques can benefit from the exploitation of the phase response of the evanescent waves, both in terms of sensitivity and reliability. In this thesis we show the versatility of Digital Holography Microscopy for the development of innovative and compact systems for quantitative phase imaging and, in particular, the implementation of through-the-objective configurations for Holographic TIR microscopy and SPRi. Advantages, issues, and applications are discussed throughout the work. At the same time, possible implementations and future perspectives are also presented with the aim to show the potential and raise interest for the development of new techniques for label-free imaging of interfaces.