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Waveguide evanescent field fluorescence microscopy allows imaging of thin films and contact regions between biological cells and their substratum. On changing the propagating waveguide mode exciting the fluorescence markers, the normalized evanescent field intensity at a specific distance from the waveguide surface is modified. This enables the determination of thin film thickness and cell-substratum separation distances. An extensive analysis of multimode ion-exchanged waveguides is presented, focusing on their applications as substrata for studying interfacial phenomena. The experimental results on Langmuir–Blodgett films are in excellent agreement with the simulations. For osteoblastic cells, close contacts were observed.