Evanescent field (EF) based fluorescence microscopy is a powerful technique to image cell- substrate contacts, solid thin films and to study molecular events occurring at the interface between biology and a solid substrate. These microscopy techniques use a thin film of light which is confined above a solid substrate to illuminate the specimen. The confinement of light provides localized fluorescence excitation of the specimen and selective excitation of fluorophores. Two microscopes have been developed based on the evanescent field: Total Internal Reflection Fluorescence Microscopy (TIRFM) and Waveguide Evanescent Field Fluorescence Microscopy (WEFFM). TIRFM is based on a simple total internal reflection from an interface between a prism and a dielectric medium (cell medium). Here, when the light is reflected back an evanescent field is generated above the prism. However, WEFFM is based on an evanescent field that can be created while a beam of light propagates in a waveguide. In both cases, the EF decays exponentially and penetrates tens of nanometers into the medium with the lower refractive index (cladding). The inhomogeneity in the illumination source results in a perfect resolution normal to the surface (z-axis) in the range of several nanometers and in very low background. These important features, plus localized fluorescence excitation of the specimen and selective excitation of fluorophores have made the evanescent based microscopy methods important imaging tools in cell biology. TIRFM and WEFFM have been applied to study dynamic biological processes which cannot be carried out with conventional or other advanced optical or even electron microscopes. The illumination of large area for imaging and allowing the use of microscope objectives with various magnifications, are some advantages which WEFFM offers over TIRFM. Grandin et al. introduced this novel imaging technique in 2006. They imaged fibroblast focal adhesions and a 10-fold improvement in the signal
