Improvement of two-dimensional structured illumination microscopy with an incoherent illumination pattern of tunable frequency.

In two-dimensional structured illumination microscopy (2D-SIM), high-resolution images with optimal optical sectioning (OS) cannot be obtained simultaneously. This tradeoff can be overcome by using a tunable-frequency 2D-SIM system and a proper reconstruction method. The goal of this work is twofold. First, we present a computational approach to reconstruct optical-sectioned images with super-resolution enhancement (OS-SR) by using a tunable SIM system. Second, we propose an incoherent tunable-frequency 2D-SIM system based on a Fresnel biprism implementation. Integration of the proposed computational method with this tunable structured illumination (SI) system results in a new 2D-SIM system that is advantageous compared to other 2D-SIM systems with comparable complexity, because it provides high-resolution OS images independent of the objective lens used, without the presence of coherent noise and without reducing the contrast of the structured pattern, as in other incoherent implementations. Evaluation of our proposed system is demonstrated with comparative studies of simulated and experimental reconstructed images to validate our theoretical findings. Our experimental results show a simultaneous improvement of the lateral resolution by a factor of 1.8× with the desired OS capability achieved in the resulting OS-SR combination image. Our experimental results also verify that our system can provide better OS capability than the commercial Zeiss ApoTome-SIM system in the investigated study.