Comparative analysis of super-resolution techniques and system development for low-cost, high-throughput gene sequencers.

The integration density of gene chips serves as a critical determinant of sequencing cost, yet its enhancement remains fundamentally constrained by the optical diffraction limit. Conventional approaches to resolution improvement often entail trade-offs that compromise other performance metrics. To address this challenge, we established a four-dimensional evaluation framework encompassing spatial resolution, optical throughput, phototoxicity, and image reconstruction speed. Three mainstream super-resolution techniques-stimulated emission depletion microscopy (STED), single-molecule localization microscopy (SMLM), and structured illumination microscopy (SIM)-were systematically compared, with SIM identified as offering the most favorable comprehensive performance. Subsequently, through optical modeling and simulation, projection-type and interference-type illumination schemes were comparatively evaluated, revealing that interference illumination based on a liquid crystal on silicon spatial light modulator (LCoS-SLM) provides superior fringe contrast and an extended field of view. A prototype system was developed following this scheme, and imaging validation was conducted across four fluorescence channels (A, T, G, C). Experimental results demonstrate that the resolution in the four channels was enhanced by factors of 1.74, 1.76, 1.92, and 1.98, respectively, relative to wide-field imaging. Furthermore, high-frequency "sequencing peak" signals corresponding to the chip period were successfully reconstructed. This study establishes a viable technological pathway toward the development of high-density, low-cost super-resolution gene sequencing systems.