Entropy-based super-resolution imaging in waveguide-based TIRF microscopy—an experimental and numerical study

Optical waveguides are transforming total internal reflection fluorescence (TIRF) microscopy by providing a larger field of view and a robust, compact, and less complex platform, which is essential for high-throughput and long-term imaging. While several methods have been explored to enhance resolution, the entropy-based super-resolution imaging (ESI) method remains underutilized despite its significant potential to improve image quality through computational means without requiring specialized hardware. In addition to limited use in the literature, existing implementations of ESI, particularly in available plugins, have notable shortcomings. These limitations often result in inaccurate conclusions, undermining the reliability and effectiveness of the technique. In this work, we first implemented our ESI algorithm and conducted simulations under various imaging conditions to identify its limitations and evaluate its suitability for TIRF microscopy. We then fabricated silicon nitride optical waveguides, coated them with TetraSpeck microspheres, and applied our ESI algorithm to analyze the experimental data. Comparative analysis with existing algorithms revealed that initial experiments using available plugins did not yield resolution enhancements and resulted in inaccurate emitter distance measurements. In contrast, our ESI algorithm effectively addressed these shortcomings, achieving resolution improvements consistent with theoretical predictions.