Wang, W.; Li, H.; Wang, Y.; Barnett, S. F. H.; Huang, Z.; Kanchanawong, P.

Ultra-high resolution 3D single-molecule localization microscopy (SMLM) traditionally requires complex dual-objective lenses (4Pi) configurations to enhance axial (z) precision through interferometry. Here we present a streamlined chip-based alternative, Silicon-assisted interferometric Localization Microscopy (SiLM), which achieves comparable performance using a single-objective lens design. By combining tunable axial structured illumination field, arising from surface-generated excitation interference, with asynchronous interferometry, SiLM enhances axial localization precision to approximately twice that of the lateral (xy), comparable to 4Pi-based methods. Additionally, SiLM provides intrinsic axial self-referencing, offering dramatically improved robustness against mechanical drift. Our method is readily implementable on standard SMLM-capable microscopes and supports a broad range of applications including dual-color imaging, extended-depth imaging, and live-cell 3D single-molecule tracking. Using SiLM, we demonstrate accurate mapping of the stratified nanoscale architecture of integrin-based focal adhesions, establishing it as a powerful and accessible method for high-precision 3D structural cell biology.