Digital holography of total internal reflection to image cell/substrate contacts

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 Fluorescence (TIRF) and Surface Plasmon Resonance imaging (SPRi) are usually the techniques of choice when high sensitivity of measurement is required. They yield information about few hundreds of nanometers from the contact surface and had proven themselves as a good tool for the characterization of thin films, cellular focal adhesions and biomolecular binding events. Moreover, they can be used either to excite fluorescent markers or in a label-free fashion. Digital Holography (DH) in microscope configuration is already a common tool to image microstructures and biological systems because of its excellent approach in obtaining intensity and quantitative phase information on a light-wave. In particular, prism-based configurations have been used to combine easily DH with total internal reflection (TIR) and SPRi [1-3]. Unfortunately, the physical constraint of the prism limits both the numerical aperture (NA) and magnification of an imaging system. Hence, it yields a poor spatial resolution compared to that achievable with optical microscopy. Moreover, resolution in prism-based imaging is mainly governed by geometrical aberrations, which distort images and, moreover, the prism limits the compatibility with conventional microscopes [4].