Systems, methods, and computer-readable storage media are disclosed for providing a structured total internal reflection fluorescence (sTIRF) imaging system providing improved out of focus blur rejection and improved image contrast. The sTIRF imaging system may be configured to illuminate a sample using two beams of light (e.g., a primary beam of light and an interfering beam of light). The interfering beam of light may be configured to create interference with respect to the primary beam of light. The sTIRF imaging system may be configured to capture a plurality of intermediate images of the sample during the illuminating, and to generate a final image of the sample based on the plurality of intermediate images. The interference caused by the two beams of light may enable the sTIRF imaging system to reject out of focus blur based on detection of whether fluorescence emissions from the sample being imaged fluctuate or remain static.