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Total internal reflection fluorescence microscopy excites labels close to a boundary using the evanescent wave accompanying total internal reflection. Illumination travels through the higher-refractive index side, typically a coverslip, at an angle above the critical angle for total internal reflection. The field penetrates the specimen side but decays with distance from the interface.

A prism or a sufficiently high-numerical aperture microscope objective supplies the required incidence. This confines excitation near the surface and reduces background from deeper fluorescent material, making it useful for surface-associated molecular events. Changing incidence angle changes the depth weighting; an intensity change can therefore reflect vertical motion as well as molecule number.^[ref]