A total internal reflection microscope for epi-fluorescence illumination observations includes an objective through which an object to be observed is illuminated by an excitation illumination light at an angle to an observation axis of the microscope. The angle is adjustable to be within the range suitable for a total internal reflection observation. The microscope also has a source of collimated excitation light. An interferometer is arranged in the optical path of the collimated excitation light and is configured to produce an interference pattern. A focusing lens system focuses the interference pattern produced by the interferometer into the back focal plane of the objective. The objective and the focusing lens system image the interference pattern produced by the interferometer into the conjugated image plane of the objective, thereby producing excitation illumination light that modulated spatially in intensity in a plane orthogonal to the observation axis of the microscope.