Waveguide evanescent field fluorescence microscopy is an evanescent field based microscopy to visualize cell-substrate contact regions and solid thin films. Despite some advantages of this method compared to other evanescent field based microscopes, non-uniform illumination source and background are reported to be the problems for producing nonuniform images. We noticed similar non-uniformities in our primary results with ultra- thin solid film and cell-substrate contact regions. We used various microscopy methods to demonstrate induced inhomogenities in the waveguide evanescent field fluorescence microscopy images by secondary patterns in the gratings, and defects in waveguides surface. We discuss their effects on the waveguide microscopy background, ultra-thin solid films, and cell-substrate contact region images. Defect-free waveguides with uniform gratings fabricated and used to image phase separated lipids monolayer LB films and cell-substrate contact regions.
