A multiple wavelength total internal reflection fluorescence (TIRF) microscopysystem has anobjective. A dispersion unit of the system comprises a high-dispersion opticalelement. Thedispersion unit receives illumination light having at least a first wavelength.lambda.1 and a secondwavelength .lambda.2, where .lambda.1 .noteq..lambda.2, and splits the illumination light into a first monochromaticbeam having the first wavelength .lambda.1 and a second monochromatic beamhaving the secondwavelength .lambda.2. The monochromatic beams are focused onto a back focalplane of theobjective, near an outer edge of the objective, at different radial distancesfrom an optical axisof the objective. The dispersion unit is rotatable in order to adjust anglesof incidence of themonochromatic beams onto an interface between a substrate and a sample to beimaged,wherein the angles of incidence are greater than the critical angle of theinterface.
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